Methods for the treatment of IL-1β related diseases

ABSTRACT

Disclosed are methods for the treatment and/or prevention of Type 2 diabetes, insulin resistance and disease states and conditions characterized by insulin resistance, obesity, hyperglycemia, hyperinsulinemia and Type 1 diabetes, comprising administering to a subject an effective amount of anti-IL-1β antibody or fragment thereof.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.11/961,764, filed Dec. 20, 2007, now U.S. Pat. No. 7,695,718, and claimsbenefit under 35 U.S.C. sctn. 119 of U.S. Provisional Application No.60/871,046, filed Dec. 20, 2006, 60/908,389, filed Mar. 27, 2007, and60/911,033, filed Apr. 10, 2007, the disclosures of which are hereinincorporated by reference in their entirety.

This application is a continuation of U.S. application Ser. No.11/961,764, filed Dec. 20, 2007, and claims benefit under 35 U.S.C. §119of U.S. Provisional Application No. 60/871,046, filed Dec. 20, 2006,60/908,389, filed Mar. 27, 2007, and 60/911,033, filed Apr. 10, 2007,the disclosures of which are herein incorporated by reference in theirentirety.

FIELD OF INVENTION

The invention relates to methods for the treatment and/or prevention ofType 2 diabetes, obesity, hyperglycemia, hyperinsulinemia, Type 1diabetes, insulin resistance and disease states and conditionscharacterized by insulin resistance. Such methods may be used to treat amammalian subject suffering from Type 2 diabetes, obesity,hyperglycemia, hyperinsulinemia, Type 1 diabetes, insulin resistance anddisease states and conditions characterized by insulin resistance or toprevent occurrence of the same in an at risk subject.

BACKGROUND OF THE INVENTION

The present disclosure is directed to methods for the treatment and/orprevention in mammals of Type 2 diabetes, obesity, hyperglycemia,hyperinsulinemia, Type 1 diabetes, insulin resistance and disease statesand conditions characterized by insulin resistance. Such methods may beused to treat a mammalian (e.g., human) subject suffering from Type 2diabetes, hyperglycemia, hyperinsulinemia, Type 1 diabetes, insulinresistance and disease states and conditions characterized by insulinresistance or to prevent occurrence of the same in an at risk subject.

Diabetes mellitus is a metabolic disorder in humans with a prevalence ofapproximately one percent in the general population (Foster, D. W.,Harrison's Principles of Internal Medicine, Chap. 114, pp. 661-678, 10thEd., McGraw-Hill, New York). The disease manifests itself as a series ofhormone-induced metabolic abnormalities that eventually lead to serious,long-term and debilitating complications involving several organ systemsincluding the eyes, kidneys, nerves, and blood vessels. Pathologically,the disease is characterized by lesions of the basement membranes,demonstrable under electron microscopy. Diabetes mellitus can be dividedinto two clinical syndromes, Type 1 and Type 2 diabetes mellitus. Type1, or insulin-dependent diabetes mellitus (IDDM), also referred to asthe juvenile onset form, is a chronic autoimmune disease characterizedby the extensive loss of beta cells in the pancreatic Islets ofLangerhans, which produce insulin. As these cells are progressivelydestroyed, the amount of secreted insulin decreases, eventually leadingto hyperglycemia (abnormally high level of glucose in the blood) whenthe amount of secreted insulin drops below the normally required bloodglucose levels. Although the exact trigger for this immune response isnot known, patients with IDDM have high levels of antibodies againstproteins expressed in pancreatic beta cells. However, not all patientswith high levels of these antibodies develop IDDM.

Type 1 diabetics characteristically show very low or immeasurable plasmainsulin with elevated glucagon. Regardless of what the exact etiologyis, most Type 1 patients have circulating antibodies directed againsttheir own pancreatic cells including antibodies to insulin, to islet ofLangerhans cell cytoplasm and to the enzyme glutamic acid decarboxylase.An immune response specifically directed against beta cells (insulinproducing cells) leads to Type 1 diabetes. The current treatment forType 1 diabetic patients is the injection of insulin, and may alsoinclude modifications to the diet in order to minimize hyperglycemiaresulting from the lack of natural insulin, which in turn, is the resultof damaged beta cells. Diet is also modified with regard to insulinadministration to counter the hypoglycemic effects of the hormone.

Type 2 diabetes (also referred to as non-insulin dependent diabetesmellitus (NIDDM), maturity onset form, adult onset form) develops whenmuscle, fat and liver cells fail to respond normally to insulin. Thisfailure to respond (called insulin resistance) may be due to reducednumbers of insulin receptors on these cells, or a dysfunction ofsignaling pathways within the cells, or both. The beta cells initiallycompensate for this insulin resistance by increasing insulin output.Over time, these cells become unable to produce enough insulin tomaintain normal glucose levels, indicating progression to Type 2diabetes. Type 2 diabetes is brought on by a combination of genetic andacquired risk factors, including a high-fat diet, lack of exercise, andaging. Greater than 90% of the diabetic population suffers from Type 2diabetes and the incidence continues to rise, becoming a leading causeof mortality, morbidity and healthcare expenditure throughout the world(Amos et al., Diabetic Med. 14:S1-85, 1997).

Type 2 diabetes is a complex disease characterized by defects in glucoseand lipid metabolism. Typically there are perturbations in manymetabolic parameters including increases in fasting plasma glucoselevels, free fatty acid levels and triglyceride levels, as well as adecrease in the ratio of HDL/LDL. As discussed above, one of theprincipal underlying causes of diabetes is thought to be an increase ininsulin resistance in peripheral tissues, principally muscle and fat.The causes of Type 2 diabetes are not well understood. It is thoughtthat both resistance of target tissues to the action of insulin anddecreased insulin secretion (“β-cell failure”) occur. Majorinsulin-responsive tissues for glucose homeostasis are liver, in whichinsulin stimulates glycogen synthesis and inhibits gluconeogenesis;muscle, in which insulin stimulates glucose uptake and glycogenstimulates glucose uptake and inhibits lipolysis. Thus, as a consequenceof the diabetic condition, there are elevated levels of glucose in theblood, which can lead to glucose-mediated cellular toxicity andsubsequent morbidity (nephropathy, neuropathy, retinopathy, etc.).Insulin resistance is strongly correlated with the development of Type 2diabetes.

Currently, there are various pharmacological approaches for thetreatment of Type 2 diabetes (Scheen et al., Diabetes Care,22(9):1568-1577, 1999). They act via different modes of action: 1)sulfonylureas (e.g., glimepiride, glisentide, sulfonylurea, AY31637)essentially stimulate insulin secretion; 2) biguanides (e.g., metformin)act by promoting glucose utilization, reducing hepatic glucoseproduction and diminishing intestinal glucose output; 3)alpha-glucosidase inhibitors (e.g., acarbose, miglitol) slow downcarbohydrate digestion and consequently absorption from the gut andreduce postprandial hyperglycemia; 4) thiazol-idinediones (e.g.,troglitazone, pioglitazone, rosiglitazone, glipizide, balaglitazone,rivoglitazone, netoglitazone, troglitazone, englitazone, AD 5075, T 174,YM 268, R 102380, NC 2100, NIP 223, NIP 221, MK 0767, ciglitazone,adaglitazone, CLX 0921, darglitazone, CP 92768, BM 152054) enhanceinsulin action, thus promoting glucose utilization in peripheraltissues; 5) glucagon-like-peptides including DPP4 inhibitors (e.g.,sitagliptin); and 6) insulin stimulates tissue glucose utilization andinhibits hepatic glucose output. The above mentioned pharmacologicalapproaches may be utilized individually or in combination therapy.However, each approach has its limitations and adverse effects. Overtime, a large percentage of Type 2 diabetic subjects lose their responseto these agents. Insulin treatment is typically instituted after diet,exercise, and oral medications have failed to adequately control bloodglucose. The drawbacks of insulin treatment are the need for druginjection, the potential for hypoglycemia, and weight gain.

IL-1β is a pro-inflammatory cytokine secreted by a number of differentcell types including monocytes and macrophages. When released as part ofan inflammatory reaction, IL-1β produces a range of biological effects,mainly mediated through induction of other inflammatory mediators suchas corticotrophin, platelet factor-4, prostaglandin E2 (PGE2), IL-6, andIL-8. IL-1β induces both local and systemic inflammatory effects throughthe activation of the IL-1 receptor found on almost all cell types. Theinterleukin-1 (IL-1) family of cytokines has been implicated in a numberof disease states. IL-1 family members include IL-1α, IL-1β, and IL-1Ra.Although related by their ability to bind to IL-1 receptors (IL-1R1 andIL-1R2), each of these cytokines is different, being expressed by adifferent gene and having a different primary amino acid sequence.Furthermore, the physiological activities of these cytokines can bedistinguished from each other. Experiments indicating the apparentinvolvement of IL-1β in diabetes have been published.

Maedler et al, J Clin Invest (2002) 110:851-860 suggested that in Type 2diabetes chronic hyperglycemia can be detrimental to pancreatic β-cells,causing impaired insulin secretion, and noted that IL-1β is aproinflammatory cytokine acting during the autoimmune process of type 1diabetes, and inhibits β cell function. In particular, they tested thehypothesis that IL-1β may mediate the deleterious effects of highglucose levels. Treatment of diabetic animals with phlorizin normalizedplasma glucose and prevented β cell expression of IL-1β. This was saidto implicate an inflammatory process in the pathogenesis ofglucotoxicity in type 2 diabetes, and they identified the IL-1β/NF-_(K)Bpathway as a target to preserve β cell mass and function in thiscondition.

Donath et al, J Mol med (2003) 81:455-470 noted the apparentsignificance of IL-1β in the pathway to apoptosis of pancreatic isletβ-cell death, leading to insulin deficiency and diabetes, and proposedanti-inflammatory therapeutic approaches designed to block β-cellapoptosis in Type 1 and 2 diabetes.

WO 2004/002512 is directed to the use of an IL-1 receptor antagonist(IL-1Ra) and/or pyrrolidine dithiocarbamate (PDTC) for the treatment orprophylaxis of type 2 diabetes. However, the frequent dosing suggestedfor therapeutic use of IL-Ra polypeptide in the treatment of Type 2diabetes (injection every 24 hours) may result in problems with patientcompliance, thereby decreasing effectiveness of this treatment modalityand/or limiting its desirability. Thus, there remains a need foreffective means to treat Type 2 diabetes, particularly those that do notrequire daily injections.

Larsen et al, New England Journal of Medicine (2007) 356:1517-1526describes the use of a recombinant IL-1 receptor antagonist (IL-1Ra,anakinra) for the treatment of type 2 diabetes mellitus. However, thedosing of 100 mg of anakinra once daily for 13 weeks may result inproblems with patient compliance, thereby decreasing effectiveness ofthis treatment modality/or limiting its desirability. Thus, thereremains a need for effective means to treat Type 2 diabetes,particularly treatment means that do not require frequent (e.g., daily)injections.

US 2005/0256197 and US 2005/0152850 are directed to a method forfacilitating metabolic control (e.g., glucose) in a subject (e.g.,subject with diabetes), comprising decreasing the level of IL-1β ingingival crevicular fluid of the subject such that the level ofcirculating TNF is decreased, particularly by using an anti-inflammatoryagent, such as an anti-inflammatory ketorolac oral rinse.

Obesity is a chronic disease that is highly prevalent and is associatednot only with a social stigma, but also with decreased life span andnumerous medical problems including adverse psychological development,dermatological disorders such as infections, varicose veins, exerciseintolerance, diabetes mellitus, insulin resistance, hypertension,hypercholesterolemia, and coronary heart disease (Rissanen et al.,British Medical Journal, 301: 835-837, 1990). Obesity is highlycorrelated with insulin resistance and diabetes in experimental animalsand humans. Indeed, obesity and insulin resistance, the latter of whichis generally accompanied by hyperinsulinemia or hyperglycemia, or both,are hallmarks of Type 2 diabetes. In addition, Type 2 diabetes isassociated with a two- to four-fold risk of coronary artery disease.Despite decades of research on these serious health problems, theetiology of obesity and insulin resistance is unknown.

Insulin resistance is associated with a number of disease states andconditions and is present in approximately 30-40% of non-diabeticindividuals. These disease states and conditions include, but are notlimited to, pre-diabetes and metabolic syndrome (also referred to asinsulin resistance syndrome). Pre-diabetes is a state of abnormalglucose tolerance characterized by either impaired glucose tolerance(IGT) or impaired fasting glucose (IFG). Patients with pre-diabetes areinsulin resistant and are at high risk for future progression to overtType 2 diabetes. Metabolic syndrome is an associated cluster of traitsthat include, but is not limited to, hyperinsulinemia, abnormal glucosetolerance, obesity, redistribution of fat to the abdominal or upper bodycompartment, hypertension, dysfibrinolysis, and a dyslipidemiacharacterized by high triglycerides, low HDL-cholesterol, and smalldense LDL particles. Insulin resistance has been linked to each of thetraits, suggesting metabolic syndrome and insulin resistance areintimately related to one another. The diagnosis of metabolic syndromeis a powerful risk factor for future development of Type 2 diabetes, aswell as accelerated atherosclerosis resulting in heart attacks, strokes,and peripheral vascular disease. Inflammatory cytokines, including IL-1,have been shown to mediate inflammation within adipose tissue whichappears to be involved in insulin resistance of adipocytes (Trayhurn etal., Br. J. Nutr. 92:347-355, 2004; Wisse, J. Am. Soc. Nephrol.15:2792-2800, 2004; Fantuzzi, J. Allergy Clin. Immunol. 115:911-919,2005; Matsuzawa, FEBS Lett. 580:2917-2921, 2006; Greenberg et al., EurJ. Clin. Invest. 32 Suppl. 3:24-34, 2002; Jager et al., Endocrinology148:241-251, 2007). Adipocytes are cells that store fat and secreteadipokines (i.e., a subset of cytokines) and are a major component ofadipose tissue. Macrophages, which are inflammatory cells and the mainproducers of the inflammatory cytokines, IL-1, TNF-α, and IL-6, alsoexist within adipose tissue, especially inflamed adipose associated withobesity (Kern et al., Diabetes 52:1779-1785, 2003). TNF-α and IL-6 havebeen known previously to desensitize adipocytes to insulin stimulation(i.e., insulin resistance).

Because of the problems with current treatments, new therapies to treatType 2 diabetes and other disease indications such as those disclosedherein are needed to replace or complement available pharmaceuticalapproaches. The present invention provides a method for treatment ofType 2 diabetes. In addition, the present invention also provides amethod for treating obesity, hyperglycemia, hyperinsulinemia, Type 1diabetes, insulin resistance and disease states and conditionscharacterized by insulin resistance. The methods disclosed hereincomprise, for example, administering an anti-IL-13 antibody or fragmentthereof. Methods that directly target the IL-1β ligand with an antibody,particularly antibodies that exhibit high affinity, provide advantagesover other potential methods of treatment, such as IL-1β receptorantagonists (e.g., IL-1Ra, Anakinra, Kineret®). The challenge for IL-1receptor antagonist-based therapeutics is the need for such therapeuticsto occupy a large number of receptors, which is a formidable task sincethese receptors are widely expressed on all cells except red blood cells(Dinarello, Curr. Opin. Pharmacol. 4:378-385, 2004). In mostimmune-mediated diseases, such as the diseases disclosed herein, theamount of IL-1β cytokine that is measurable in body fluids or associatedwith activated cells is relatively low. Thus, a method of treatmentand/or prevention that directly targets the IL-1β ligand is a superiorstrategy, particularly when administering an IL-1β antibody with highaffinity.

SUMMARY OF THE INVENTION

The present disclosure is directed to methods for the treatment and/orprevention in mammals of Type 2 diabetes, obesity, hyperglycemia,hyperinsulinemia, decreased insulin production, Type 1 diabetes, insulinresistance and/or disease states and conditions characterized by insulinresistance. Such methods may be used to treat a mammalian (e.g., human)subject suffering from or at risk for diabetes, obesity, hyperglycemia,hyperinsulinemia, decreased insulin production, insulin resistanceand/or disease states and conditions characterized by insulinresistance. The methods also may be used to prevent the occurrence ofType 2 diabetes, Type 1 diabetes, obesity, hyperglycemia,hyperinsulinemia, decreased insulin production, insulin resistance anddisease states and conditions characterized by insulin resistance in anat risk subject.

In one aspect, the invention is a method of treating in a human, adisease or condition selected from the group consisting of Type 2diabetes, hyperglycemia, hyperinsulinemia, obesity, decreased insulinproduction, Type 1 diabetes, and insulin resistance, the methodcomprising administering an anti-IL-1β antibody or fragment thereof tothe human. In a preferred embodiment, the disease or condition isselected from the group consisting of Type 2 diabetes, hyperglycemia,hyperinsulinemia, obesity, decreased insulin production, and insulinresistance. Preferably, the disease or condition is Type 2 diabetes,obesity, decreased insulin production, or insulin resistance. Morepreferably the disease or condition is Type 2 diabetes, obesity, orinsulin resistance. Most preferably the disease or condition is Type 2diabetes. In one embodiment, the method does not augment acardiovascular disease or condition. In certain embodiments, theantibody or fragment is used to treat two or more of the aforementioneddiseases or conditions in the same patient (e.g., human subject).

In another aspect, the invention provides a method for treating orpreventing a disease or condition by administering an anti-IL-1βantibody or fragment, wherein the disease or condition is pre-diabetes,dyslipidemia, hyperlipidemia, hypertension, metabolic syndrome orsickness behavior. In yet another aspect, the method reduces or preventsin a human a complication or condition associated with Type 2 diabetesselected from the group consisting of retinopathy, renal failure,cardiovascular disease, and wound healing, the method comprisingadministering an anti-IL-1β antibody or fragment thereof to the human.In certain embodiments, the antibody or fragment is used to treat two ormore of the aforementioned diseases or conditions in the same patient(e.g., human subject). In another embodiment, the antibody or fragmentis used to treat renal failure (e.g., renal disease) that may resultfrom a condition other than Type 2 diabetes. In yet another embodiment,the antibody or fragment is used to decrease the level of C-reactiveprotein (CRP) in a subject exhibiting elevated levels of CRP.

In another aspect, the invention provides IL-1β antibodies or antibodyfragments thereof for use in treating or preventing a disease orcondition as disclosed herein. In a further aspect, the inventionprovides IL-1β antibodies or antibody fragments thereof for use intreating or preventing a disease or conditions selected from the groupconsisting of Type 2 diabetes, obesity, decreased insulin production,and insulin resistance. In yet another aspect, the invention providesIL-1β antibodies or antibody fragments thereof for use in treating orpreventing Type 2 diabetes.

In another aspect, the invention provides pharmaceutical compositionscomprising IL-1β antibodies or antibody fragments thereof and optionallyat least one pharmaceutically acceptable excipient for use in treatingor preventing a disease or condition as disclosed herein. In a furtheraspect, the invention provides pharmaceutical compositions comprisingIL-1β antibodies or antibody fragments thereof and optionally at leastone pharmaceutically acceptable excipient for use in treating orpreventing a disease or conditions selected from the group consisting ofType 2 diabetes, obesity, decreased insulin production, and insulinresistance. In yet another aspect, the invention provides pharmaceuticalcompositions comprising IL-1β antibodies or antibody fragments thereofand optionally at least one pharmaceutically acceptable excipient foruse in treating or preventing Type 2 diabetes.

The anti-IL-1β antibodies or antibody fragments used in the methods ofthe invention generally bind to IL-1β with high affinity. In preferredembodiments, the antibody or antibody fragment binds to IL-1β with adissociation constant of about 10 nM or less, about 5 nM or less, about1 nM or less, about 500 pM or less, about 250 pM or less, about 100 pMor less, about 50 pM or less, or about 25 pM or less. In particularlypreferred embodiments, the antibody or antibody fragment binds to humanIL-1β with a dissociation constant of about 100 pM or less, about 50 pMor less, about 10 pM or less, about 5 pM or less, about 3 pM or less,about 1 pM or less, about 0.75 pM or less, about 0.5 pM or less, about0.3 pM or less, about 0.2 pM or less, or about 0.1 pM or less. Inparticularly preferred embodiments, the antibody or antibody fragmentbinds to human IL-1β with a dissociation constant of about 10 pM orless.

In another aspect of the invention, the anti-IL-1β antibody or antibodyfragment is a neutralizing antibody. In another aspect, the anti-IL-1βantibody or antibody fragment binds to an IL-1β epitope such that thebound antibody or fragment substantially permits the binding of IL-1β toIL-1 receptor I (IL-1RI). In another aspect, the anti-IL-1β antibody orantibody fragment binds to IL-1β, but does not substantially prevent thebound IL-1β from binding to IL-1 receptor I (IL-1RI). In another aspect,the antibody or antibody fragment does not detectably bind to IL-1α,IL-1R or IL-1Ra. In yet another aspect of the invention, the antibody orantibody fragment binds to an epitope contained in the sequenceESVDPKNYPKKKMEKRFVFNKIE (SEQ ID NO: 1). In yet another aspect of theinvention, the antibody or antibody fragment binds to an epitopeincorporating Glu64 of IL-1β. In yet another aspect of the invention,the antibody or antibody fragment binds to amino acids 1-34 of the Nterminus of IL-1β. Preferably, the antibody or antibody fragment ishuman engineered, humanized or human.

In another aspect, the invention provides a method of treating a humandisplaying symptoms of, or at risk for, developing any of theaforementioned diseases or conditions (e.g., Type 1 diabetes, Type 2diabetes, hyperglycemia, hyperinsulinemia, obesity, decreased insulinproduction, insulin resistance), the method comprising administering ananti-IL-1β antibody or fragment thereof to the human in one or moredoses.

In another aspect of the invention, a method is provided for treating ina human, a disease or condition selected from the group consisting ofType 1 diabetes, Type 2 diabetes, hyperglycemia, hyperinsulinemia,obesity, decreased insulin production, and insulin resistance, themethod comprising administering an anti-IL-1β antibody or fragmentthereof to the human, wherein administration of an initial dose of theIL-1β antibody or antibody fragment is followed by the administration ofone or more subsequent doses. In one embodiment, administration of aninitial dose of the antibody or antibody fragment is followed by theadministration of two or more subsequent doses. In another embodiment,administration of an initial dose of the antibody or antibody fragmentis followed by the administration of one or more subsequent doses, andwherein said one or more subsequent doses are in an amount that isapproximately the same or less than the initial dose. In anotherembodiment, administration of an initial dose of the antibody orantibody fragment is followed by the administration of one or moresubsequent doses, and wherein at least one of the subsequent doses is inan amount that is more than the initial dose.

In one embodiment, two or more, three or more, four or more, five ormore, six or more, seven or more, eight or more, nine or more, ten ormore or eleven or more subsequent doses of the antibody areadministered. In another embodiment administration of the initial doseand each one or more subsequent doses are separated from each other byan interval of at least about two weeks, at least about three weeks, atleast about one month, at least about two months, at least about threemonths, at least about four months, at least about five months, at leastabout six months, at least about seven months, at least about eightmonths, at least about nine months, at least about ten months, at leastabout eleven months, or at least about twelve months.

In another embodiment, the antibody or fragment is administered in oneor more doses of 5 mg/kg or less of antibody or fragment, 3 mg/kg orless of antibody or fragment, 2 mg/kg or less of antibody or fragment, 1mg/kg or less of antibody or fragment, 0.75 mg/kg or less of antibody orfragment, 0.5 mg/kg or less of antibody or fragment, 0.3 mg/kg or lessof antibody or fragment, 0.1 mg/kg or less of antibody or fragment, or0.03 mg/kg or less of antibody or fragment. Preferably, in each of theaforementioned embodiments, the antibody or fragment is administered inone or more doses of at least 0.01 mg/kg of antibody or fragment, atleast, 0.03 mg/kg of antibody or fragment, at least 0.05 mg/kg ofantibody or fragment, or at least 0.09 mg/kg of antibody or fragment.The above dosage amounts refer to mg (antibody or fragment)/kg (weightof the individual to be treated).

In another embodiment, the initial dose and one or more subsequent dosesof antibody or fragment are each from about 0.01 mg/kg to about 10 mg/kgof antibody, from about 0.05 to about 5 mg/kg of antibody, from about0.05 mg/kg to about 3 mg/kg of antibody, from about 0.1 mg/kg to about 3mg/kg of antibody, from about 0.1 mg/kg to about 1 mg/kg of antibody,from about 0.1 mg/kg to about 0.5 mg/kg of antibody, from about 0.3mg/kg to about 5 mg/kg of antibody, from about 0.3 mg/kg to about 3mg/kg of antibody, from about 0.3 mg/kg to about 1 mg/kg of antibody,from about 0.5 mg/kg to about 5 mg/kg of antibody, from about 0.5 mg/kgto about 3 mg/kg of antibody, from about 0.5 mg/kg to about 1 mg/kg ofantibody, from about 1 mg/kg to about 5 mg/kg of antibody, or from about1 mg/kg to about 3 mg/kg of antibody. In certain embodiments, two ormore, three or more, four or more, five or more, six or more, seven ormore, eight or more, nine or more, ten or more or eleven or moresubsequent doses of the antibody are administered. The above dosageamounts refer to mg (antibody or fragment)/kg (weight of the individualto be treated). The same applies hereinafter if a dosage amount ismentioned.

In another aspect, the invention provides a method of treating in ahuman, a disease or condition selected from the group consisting of Type1 diabetes, Type 2 diabetes, hyperglycemia, hyperinsulinemia, obesity,decreased insulin production, and insulin resistance, the methodcomprising administering a therapeutically effective amount of ananti-IL-1β antibody or fragment thereof to the human as an initial doseof about 5 mg/kg or less of antibody or fragment and a plurality ofsubsequent doses of antibody or fragment in an amount about the same orless than the initial dose, wherein the subsequent doses are separatedby an interval of time of at least 2 weeks.

In another aspect, the invention provides a method of treating in ahuman, a disease or condition selected from the group consisting of Type1 diabetes, Type 2 diabetes, hyperglycemia, hyperinsulinemia, obesity,decreased insulin production, and insulin resistance, the methodcomprising administering a therapeutically effective amount of ananti-IL-1β antibody or fragment thereof to the human as an initial doseof about 3 mg/kg or less of antibody or fragment and a plurality ofsubsequent doses of antibody or fragment in an amount about the same orless than the initial dose, wherein the subsequent doses are separatedby an interval of time of at least 2 weeks.

In another aspect, the invention provides a method of treating in ahuman, a disease or condition selected from the group consisting of Type1 diabetes, Type 2 diabetes, hyperglycemia, hyperinsulinemia, obesity,decreased insulin production, and insulin resistance, the methodcomprising administering a therapeutically effective amount of ananti-IL-1β antibody or fragment thereof to the human as an initial doseof about 1 mg/kg or less of antibody or fragment and a plurality ofsubsequent doses of antibody or fragment in an amount about the same orless than the initial dose, wherein the subsequent doses are separatedby an interval of time of at least 2 weeks.

In another aspect, the invention provides a method of treating in ahuman, a disease or condition selected from the group consisting of Type1 diabetes, Type 2 diabetes, hyperglycemia, hyperinsulinemia, obesity,decreased insulin production, and insulin resistance, the methodcomprising administering a therapeutically effective amount of ananti-IL-1β antibody or fragment thereof to the human as an initial doseof about 0.5 mg/kg or less of antibody or fragment and a plurality ofsubsequent doses of antibody or fragment in an amount about the same orless than the initial dose, wherein the subsequent doses are separatedby an interval of time of at least 2 weeks.

In another aspect, the invention provides a method of treating in ahuman, a disease or condition selected from the group consisting of Type1 diabetes, Type 2 diabetes, hyperglycemia, hyperinsulinemia, obesity,decreased insulin production, and insulin resistance, the methodcomprising administering a therapeutically effective amount of ananti-IL-1β antibody or fragment thereof to the human as an initial doseof about 0.3 mg/kg or less of antibody or fragment and a plurality ofsubsequent doses of antibody or fragment in an amount about the same orless than the initial dose, wherein the subsequent doses are separatedby an interval of time of at least 2 weeks.

In another aspect, the invention provides a method of treating in ahuman, a disease or condition selected from the group consisting of Type1 diabetes, Type 2 diabetes, hyperglycemia, hyperinsulinemia, obesity,decreased insulin production, and insulin resistance, the methodcomprising administering a therapeutically effective amount of ananti-IL-1β antibody or fragment thereof to the human as an initial doseof about 0.1 mg/kg or less of antibody or fragment and a plurality ofsubsequent doses of antibody or fragment in an amount about the same orless than the initial dose, wherein the subsequent doses are separatedby an interval of time of at least 2 weeks.

Preferably, in the aforementioned embodiments wherein the antibody orfragment is administered as an initial dose and a plurality ofsubsequent doses, the dose of antibody or fragment is at least 0.01mg/kg of antibody or fragment, at least, 0.03 mg/kg of antibody orfragment, at least 0.05 mg/kg of antibody or fragment, or at least 0.09mg/kg of antibody or fragment.

In yet another aspect of the invention, the antibody or fragment isadministered as a fixed dose, independent of a dose per subject weightratio. In one embodiment, the antibody or fragment is administered inone or more fixed doses of 1000 mg or less of antibody or fragment, 750mg or less of antibody or fragment, 500 mg or less of antibody orfragment, 250 mg or less of antibody or fragment, 100 mg or less ofantibody or fragment, or about 25 mg or less of antibody or fragment. Inanother embodiment, the antibody or fragment is administered in one ormore fixed doses of at least about 1 mg of antibody or fragment, atleast about 5 mg of antibody or fragment, or at least about 10 mg ofantibody or fragment.

In certain embodiments, the fixed dose is from about 1 mg to about 10mg, about 1 mg to about 25 mg, about 10 mg to about 25 mg, about 10 mgto about 50 mg, about 10 mg to about 100 mg, about 25 mg to about 50 mg,about 25 mg to about 100 mg, about 50 mg to about 100 mg, about 50 mg toabout 150 mg, about 100 mg to about 150 mg, about 100 mg to about 200mg, about 150 mg to about 200 mg, about 150 mg to about 250 mg, about200 mg to about 250 mg, about 200 mg to about 300 mg, about 250 mg toabout 300 mg, about 250 mg to about 500 mg, about 300 mg to about 400mg, about 400 mg to about 500 mg, about 400 mg to about 600 mg, about500 mg to about 750 mg, about 600 mg to about 750 mg, about 700 mg toabout 800 mg, about 750 mg to about 1000 mg. In a preferred embodiment,the fixed dose is selected from the group consisting of about 1 mg toabout 10 mg, about 1 mg to about 25 mg, about 10 mg to about 25 mg,about 10 mg to about 100 mg, about 25 mg to about 50 mg, about 50 mg toabout 100 mg, about 100 mg to about 150 mg, about 150 mg to about 200mg, about 200 mg to about 250 mg.

In another aspect, the invention provides a method of treating in ahuman, a disease or condition selected from the group consisting of Type1 diabetes, Type 2 diabetes, hyperglycemia, hyperinsulinemia, obesity,decreased insulin production, and insulin resistance, the methodcomprising administering a therapeutically effective amount of ananti-IL-1β antibody or fragment thereof to the human, whereinadministration of an initial dose of the antibody or antibody fragmentis followed by the administration of one or more subsequent doses, andwherein the plasma concentration of said antibody or antibody fragmentin the human is permitted to decrease below a level of about 0.1 ug/mLfor a period of time greater than about 1 week and less than about 6months between administrations during a course of treatment with saidinitial dose and one or more subsequent doses. In one embodiment, theplasma concentration of said antibody or antibody fragment is permittedto decrease below a level of about 0.07 ug/mL, about 0.05 ug/mL, about0.03 ug/mL or about 0.01 ug/mL for a period of time greater than about 1week and less than about 5 months, about 4 months, about 3 months, about2 months, about 1 month, about 3 weeks, or about 2 weeks betweenadministrations. In one embodiment, these plasma values refer to valuesobtained for an individual that is treated with the antibody of fragmentin accordance with the invention. In one embodiment, such an individualmay be a patient suffering from one of the diseases mentionedhereinafter such as Type 2 diabetes.

The invention contemplates that an anti-IL-1β antibody or fragment usedin accordance with the methods herein may be administered in any of theaforementioned dose amounts, numbers of subsequent administrations, anddosing intervals between administrations, and that any of the discloseddose amounts, numbers of subsequent administrations, and dosingintervals between administrations may be combined with each other inalternative regimens to modulate the therapeutic benefit. In certainembodiments, the one or more subsequent doses are in an amount that isapproximately the same or less than the first dose administered. Inanother embodiment, the one or more subsequent doses are in an amountthat is approximately more than the first dose administered. Preferablythe anti-IL-1β antibody or fragment is administered by subcutaneous,intramuscular or intravenous injection. The invention contemplates thateach dose of antibody or fragment may be administered at one or moresites.

In another aspect, the invention provides a method of treating orpreventing a disease or condition in a human using an anti-IL-1βantibody or antibody fragment, wherein the disease or condition is Type2 diabetes, and wherein a dose of the antibody or fragment is sufficientto achieve at least a 0.5, at least a 1.0, at least a 1.5, at least a2.0, at least a 2.5, or at least a 3.0 percentage point improvement inhemoglobin A1c. In one embodiment, these parameter values refer tovalues obtained for an individual that is treated with the antibody offragment in accordance with the invention. In one embodiment, such anindividual may be a patient suffering from one of the diseases mentionedhereinafter such as Type 2 diabetes.

In a preferred embodiment, the improvement in hemoglobin A1c issufficient to meet regulatory guidelines for approval of therapeuticagents in Type 2 diabetes treatment. Assay methods for determination ofhemoglobin A1c are well known in the art. The invention contemplatesthat the dose of antibody or fragment sufficient to achieve theimprovement in hemoglobin A1c, may comprise any of the aforementioneddose amounts, numbers of subsequent administrations, and dosingintervals between administrations, as well as any combination of doseamounts numbers of subsequent administrations, and dosing intervalsbetween administrations antibody or fragment described herein. Further,the improvement in hemoglobin A1c may be at a time-point at least about1 month, about 2 months, about 3 months, about 4 months, or about 5months, and preferably about 6 months or more, about 7 months or more,about 8 months or more, about 9 months or more, about 10 months or more,about 11 months or more, or about 12 months or more following an initialadministration of one or more doses of antibody or fragment.

In another aspect of the aforementioned methods for treating orpreventing Type 2 diabetes, the method is sufficient to achieve at leastone of the following modifications: reduction in fasting blood sugarlevel, decrease in insulin resistance, reduction of hyperinsulinemia,improvement in glucose tolerance, reduction in C-reactive peptide (CRP),increased insulin production and reduction of hyperglycemia, reductionin the need for diabetes medication, reduction in BMI, change inglucose/insulin C-peptide AUC, reduction in urine glucose level,reduction in acute phase reactants, decrease in serum lipids withimprovement in the lipid profile with respect to cardiovascular risk.Assay methods for determining any of the above modifications are wellknown in the art. Further, the invention contemplates that achievementof one of the aforementioned modifications may be at a time-point atleast about 1 month, about 2 months, about 3 months, about 4 months, orabout 5 months, and preferably at least about 6 months or more, about 7months or more, about 8 months or more, about 9 months or more, about 10months or more, about 11 months or more, or about 12 months or morefollowing an initial administration of one or more doses of antibody orfragment.

In another aspect of the invention, the method provided herein reducesor prevents a complication or condition associated Type 2 diabetesselected from the group consisting of retinopathy, renal failure,cardiovascular disease, and wound healing, the method comprisingadministering an anti-IL-1β antibody or fragment thereof to the human.In one embodiment, the complication or condition is cardiovasculardisease, and wherein said cardiovascular disease is atherosclerosis orperipheral vascular disease. In another embodiment, the complication orcondition is wound healing, and wherein said wound healing condition isdiabetic ulcer. In another aspect, the method prevents or delays endstage renal disease or diabetic neuropathy. In one embodiment, theanti-IL-1β antibody or fragment is administered in combination with atleast one other medically accepted treatment for the disease, conditionor complication. In another embodiment, the at least one other medicallyaccepted treatment for the disease, condition or complication is reducedor discontinued, while treatment with the anti-IL-1β antibody orfragment is maintained at a constant dosing regimen. In anotherembodiment, the at least one other medically accepted treatment for thedisease, condition or complication is reduced or discontinued, andtreatment with the anti-IL-1β antibody or fragment is reduced. Inanother embodiment, the at least one other medically accepted treatmentfor the disease, condition or complication is reduced or discontinued,and treatment with the anti-IL-1β antibody or fragment is increased. Inyet another embodiment, the at least one other medically acceptedtreatment for the disease, condition or complication is maintained andtreatment with the anti-IL-1β antibody or fragment is reduced ordiscontinued. In yet another embodiment, the at least one othermedically accepted treatment for the disease, condition or complicationand treatment with the anti-IL-1β antibody or fragment are reduced ordiscontinued.

In another aspect of the invention, a method of reducing the amount ofC-reactive protein in a subject is provided, the method comprisingadministering an anti-IL-1β antibody or fragment thereof to the subject.In one embodiment, the antibody or antibody fragment is administered inone or more doses of 1 mg/kg or less of antibody or fragment, 0.75 mg/kgor less of antibody or fragment, 0.5 mg/kg or less of antibody orfragment, 0.3 mg/kg or less of antibody or fragment, 0.1 mg/kg or lessof antibody or fragment, or 0.03 mg/kg or less of antibody or fragment.Preferably, the antibody or fragment is administered in one or moredoses of at least 0.01 mg/kg of antibody or fragment, at least, 0.03mg/kg of antibody or fragment, at least 0.05 mg/kg of antibody orfragment, or at least 0.09 mg/kg of antibody or fragment. In anotherembodiment, the antibody or fragment is administered as one or morefixed doses, independent of a dose per subject weight ratio, of 500 mgor less of antibody or fragment, 250 mg or less of antibody or fragment,100 mg or less of antibody or fragment, or about 25 mg or less ofantibody or fragment. Preferably, the antibody or fragment isadministered in one or more fixed doses of at least about 1 mg ofantibody or fragment, at least about 5 mg of antibody or fragment, or atleast about 10 mg of antibody or fragment. In another embodiment, theantibody or antibody fragment binds to IL-1β with a dissociationconstant of about 500 pM or less, 250 pM or less, about 100 pM or less,about 50 pM or less, or about 25 pM or less, about 10 pM or less, about5 pM or less, about 3 pM or less, about 1 pM or less, about 0.75 pM orless, about 0.5 pM or less, about 0.3 pM or less, about 0.2 pM or less,or about 0.1 pM or less. In another embodiment, administration of aninitial dose is followed by administration of one or more subsequentdoses, separated from each other by an interval of at least about twoweeks, at least about three weeks, at least about one month, at leastabout two months, at least about three months, at least about fourmonths, at least about five months, at least about six months, at leastabout seven months, at least about eight months, at least about ninemonths, at least about ten months, at least about eleven months, or atleast about twelve months. In another embodiment, said method ofreducing the amount of C-reactive protein in a subject is provided,wherein the subject is suffering from a renal disease (e.g., chronicrenal disease, renal failure). In another embodiment, the subject issuffering from Type 2 diabetes, Type 1 diabetes, obesity, hyperglycemia,hyperinsulinemia, decreased insulin production, insulin resistanceand/or disease states and conditions characterized by insulinresistance. In another embodiment, the subject is suffering from adisease or condition of pre-diabetes, dyslipidemia, hyperlipidemia,hypertension, metabolic syndrome or sickness behavior. The above dosageamounts refer to mg (antibody or fragment)/kg (weight of the individualto be treated). Administration of the antibodies or fragments with theaforementioned dissociation constants may be performed according to anyof the aforementioned dose amounts and dosing intervals (whenadministering two or more doses).

In another aspect, methods provided herein are in conjunction with atleast one additional treatment method, said additional treatment methodcomprising administering at least one pharmaceutical compositioncomprising an active agent other than an IL-1β antibody or fragment. Inyet another aspect, the methods prevent or delay the need for at leastone additional treatment method, said additional treatment methodcomprising administering at least one pharmaceutical compositioncomprising an active agent other than an IL-1β antibody or fragment. Instill another aspect, the methods reduce the amount, frequency orduration of at least one additional treatment method, said additionaltreatment method comprising administering at least one pharmaceuticalcomposition comprising an active agent other than an IL-1β antibody orfragment. In one embodiment, the at least one pharmaceutical compositioncomprising an active agent other than an IL-1β antibody or fragment isselected from the group consisting of a sulfonylurea, a meglitinide, abiguanide, an alpha-glucosidase inhibitor, a thiazolidinedione, aglucagon-like peptide, and insulin. In another embodiment, the activeagent is a sulfonylurea. In another embodiment, the active agent is ameglitinide. In another embodiment, the active agent is a biguanide. Inanother embodiment, the active agent is an alpha-glucosidase inhibitor.In another embodiment, the active agent is a thiazolidinedione. Inanother embodiment, the active agent is a glucagon-like peptide. Inanother embodiment, the active agent is insulin. In another embodiment,the at least one pharmaceutical composition comprising an active agent,comprises two active agents. In one embodiment, the two active agentsare a sulfonylurea and a biguanide. In another embodiment, the twoactive agents are a thiazolidinedione and a biguanide. In yet anotherembodiment, treatment with the at least one active agent is maintained.In another embodiment, treatment with the at least one active agent isreduced or discontinued, while treatment with the anti-IL-1β antibody orfragment is maintained at a constant dosing regimen. In anotherembodiment, treatment with the at least one active agent is reduced ordiscontinued and treatment with the anti-IL-1β antibody or fragment isreduced. In another embodiment, treatment with the at least one activeagent is reduced or discontinued, and treatment with the anti-IL-1βantibody or fragment is increased. In yet another embodiment, treatmentwith the at least one active agent is maintained and treatment with theanti-IL-1β antibody or fragment is reduced or discontinued. In yetanother embodiment, treatment with the at least one active agent andtreatment with the anti-IL-1β antibody or fragment are reduced ordiscontinued.

In another aspect, the invention provides a method of treating in ahuman, a disease or condition selected from the group consisting of Type1 diabetes, Type 2 diabetes, hyperglycemia, hyperinsulinemia, obesity,decreased insulin production, and insulin resistance, the methodcomprising administering a therapeutically effective amount of ananti-IL-1β antibody or fragment thereof to the human, whereinadministration of an initial dose of the antibody or antibody fragmentis followed by the administration of one or more subsequent doses, andwherein the plasma concentration of said antibody or antibody fragmentin the human is maintained at a level of at least about 0.03 ug/mL, atleast about 0.05 ug/mL, at least about 0.08 ug/mL, at least about 0.1ug/mL, at least about 0.15 ug/mL, at least about 0.2 ug/mL, at leastabout 0.25 ug/mL, at least about 0.3 ug/mL, at least about 0.4 ug/mL, atleast about 0.5 ug/mL, at least about 0.6 ug/mL, at least about 0.8ug/mL, at least about 1 ug/mL, at least about 1.5 ug/mL, at least about2 ug/mL, at least about 3 ug/mL, at least about 4 ug/mL, or at leastabout 5 ug/mL during a course of treatment with said initial dose andone or more subsequent doses. In one embodiment, these plasma valuesrefer to values obtained for an individual that is treated with theantibody of fragment in accordance with the invention. In oneembodiment, such an individual may be a patient suffering from one ofthe diseases mentioned hereinafter such as Type 2 diabetes.

In another aspect, the invention provides a method of treating in ahuman, a disease or condition selected from the group consisting of Type1 diabetes, Type 2 diabetes, hyperglycemia, hyperinsulinemia, obesity,decreased insulin production, and insulin resistance, the methodcomprising administering a therapeutically effective amount of ananti-IL-1β antibody or fragment thereof to the human, whereinadministering the anti-IL-1β antibody or fragment thereof to the humanresults in a decrease in the production of one or more gene productsselected from the group consisting of leptin, resistin, visfatin,RANTES, IL-6, MCP-1, PAI-1, acylation-stimulating protein, SAA3,Pentraxin-3, macrophage migration inhibition factor, IL-1RA, IL-12, IL-8and TNF-α. In one embodiment, administering the anti-IL-1β antibody orfragment thereof to the human results in a decrease in the production ofone or more gene products selected from the group consisting of leptin,resistin, and visfatin. In another embodiment, administering theanti-IL-1β antibody or fragment thereof to the human results in adecrease in the production of one or more gene products selected fromthe group consisting of MCP-1, RANTES, IL-6, TNF-α, and Pentraxin-3. Inyet another embodiment, said decrease in the production of one or moregene products is from adipose tissue. In yet another embodiment, saiddecrease is detected in the blood of the human.

In another aspect, the invention provides a method of treating in ahuman, a disease or condition selected from the group consisting of Type1 diabetes, Type 2 diabetes, hyperglycemia, hyperinsulinemia, obesity,decreased insulin production, and insulin resistance, the methodcomprising administering a therapeutically effective amount of ananti-IL-1β antibody or fragment thereof to the human, whereinadministering the anti-IL-1β antibody or fragment thereof to the humanresults in an increase in the production of adiponectin. In oneembodiment, said increase in the production of adiponectin is fromadipose tissue. In another embodiment, said increase is detected in theblood of the human.

In another aspect, the invention provides a method of treating in ahuman, a disease or condition selected from the group consisting of Type1 diabetes, Type 2 diabetes, hyperglycemia, hyperinsulinemia, obesity,decreased insulin production, and insulin resistance, the methodcomprising administering a therapeutically effective amount of ananti-IL-1β antibody or fragment thereof to the human, wherein theantibody or fragment thereof has a lower IC₅₀ than an IL-1β receptorantagonist in a human whole blood IL-1β inhibition assay that measuresIL-1β induced production of IL-8. In one embodiment, the antibody orfragment has an IC₅₀ that is less than about 90%, 80%, 70%, 60%, 50% ofthe IC₅₀ of an IL-1β receptor antagonist in a human whole blood IL-1βinhibition assay that measures IL-1β induced production of IL-8. In afurther embodiment, the antibody or fragment has an IC₅₀ that is lessthan about 40%, 30%, 20%, 10% of the IC₅₀ of an IL-1β receptorantagonist in a human whole blood IL-1β inhibition assay that measuresIL-1β induced production of IL-8. In a preferred embodiment, theantibody or fragment has an IC₅₀ that is less than about 8%, 5%, 4%, 3%,2%, 1% of the IC₅₀ of an IL-1β receptor antagonist in a human wholeblood IL-1β inhibition assay that measures IL-1β induced production ofIL-8. In one embodiment, the IL-1β receptor antagonist is anakinra(i.e., Kineret®).

In another aspect, the invention provides a method of treating in ahuman, a disease or condition selected from the group consisting of Type1 diabetes, Type 2 diabetes, hyperglycemia, hyperinsulinemia, obesity,decreased insulin production, and insulin resistance, the methodcomprising administering a therapeutically effective amount of ananti-IL-1β antibody or fragment thereof to the human, wherein theantibody or fragment thereof provides in vivo inhibition of IL-1βstimulated release of IL-6 in mice compared to a control antibody usingan assay that is described by Economides et al., Nature Med., 9:47-52(2003) which is incorporated by reference. In one embodiment theantibody or fragment provides in vivo inhibition of IL-1β stimulatedrelease of IL-6 in mice of at least about 10%, 20%, 30%, 40%, 50%compared to the control antibody. In a further embodiment, the antibodyor fragment provides in vivo inhibition of IL-1β stimulated release ofIL-6 in mice of at least about 60%, 70%, 80%, 90%, 95% compared to thecontrol antibody. In one embodiment, the control antibody is an isotypecontrol antibody.

In another aspect, the invention provides a method of treating in ahuman, a disease or condition selected from the group consisting of Type1 diabetes, Type 2 diabetes, hyperglycemia, hyperinsulinemia, obesity,decreased insulin production, and insulin resistance, the methodcomprising administering a therapeutically effective amount of ananti-IL-1β antibody or fragment thereof to the human, wherein theantibody or fragment thereof inhibits Staphylococcus epidermidis inducedcytokine production in human whole blood compared to a control where noantibody is used. In one embodiment the antibody or fragment provides agreater level of inhibition of Staphylococcus epidermidis inducedcytokine production in human whole blood by at least about 10%, 20%,30%, 40%, 50% compared to the control. In a further embodiment, theantibody or fragment provides a greater level of inhibition ofStaphylococcus epidermidis induced cytokine production in human wholeblood by at least about 60%, 70%, 80%, 90%, 95% compared to the control.In one embodiment, the inhibited cytokines are IL-1β, IL-1a, IL-6, IL-8,IL-1Ra, TNFα or IFNγ.

In another aspect, the invention discloses the use of an anti-IL-1βantibody or fragment thereof which as a lower IC₅₀ than an IL-1βreceptor antagonist in a human whole blood IL-1β inhibition assay thatmeasures IL-1β induced production of IL-8, in the manufacture of acomposition for use in the treatment of Type 1 diabetes, Type 2diabetes, hyperglycemia, hyperinsulinemia, obesity, decreased insulinproduction, and insulin resistance. In one embodiment, the IL-1βreceptor antagonist is anakinra (i.e., Kineret®)

In another aspect of the invention, the use of the IL-1β antibodies orbinding fragments is contemplated in the manufacture of a medicament fortreating or preventing a disease or condition as disclosed herein. Inany of the uses, the medicament can be coordinated with treatment usinga second active agent. In another embodiment of the invention, the useof a synergistic combination of an antibody of the invention forpreparation of a medicament for treating a patient exhibiting symptomsof at risk for developing a disease or condition as disclosed herein,wherein the medicament is coordinated with treatment using a secondactive agent is contemplated. In yet another related embodiment, thecomposition is provided wherein the second active agent is anotherantibody, a growth factor, a cytokine or insulin. Embodiments of any ofthe aforementioned uses are contemplated wherein the amount of the IL-1βbinding antibody or fragment in the medicament is at a dose effective toreduce the dosage of second active agent required to achieve atherapeutic effect.

In yet another aspect of the invention, an article of manufacture isprovided, comprising a container, a composition within the containercomprising an anti-IL-1β antibody or fragment thereof, and a packageinsert containing instructions to administer the antibody or fragment toa human in need of treatment according to the aforementioned methods ofthe invention. In one embodiment, the container further comprises apharmaceutically suitable carrier, excipient or diluent. In a relatedembodiment, the composition within the container further comprises asecond active agent. In yet another related embodiment, the compositionis provided wherein the second active agent is another antibody, agrowth factor, a cytokine or insulin.

Kits are also contemplated by the present invention. In one embodiment,a kit comprises a therapeutically or prophylactically effective amountof an anti-IL-1β antibody or fragment, packaged in a container, such asa vial or bottle, and further comprising a label attached to or packagedwith the container, the label describing the contents of the containerand providing indications and/or instructions regarding use of thecontents of the container for treatment or prevention of a disease orcondition according to the aforementioned methods of the invention. Inone embodiment, the container further comprises a pharmaceuticallysuitable carrier, excipient or diluent. In a related embodiment, thecontainer further contains a second active agent. In yet another relatedembodiment, the second active agent comprises another antibody, a growthfactor, a cytokine or insulin.

In one embodiment, the article of manufacture, kit or medicament is forthe treatment or prevention of a disease or condition in a human, saiddisease or condition selected from the group consisting of Type 1diabetes, Type 2 diabetes, hyperglycemia, hyperinsulinemia, obesity,decreased insulin production, and insulin resistance. In one preferredembodiment, the disease or condition is selected from the groupconsisting of Type 2 diabetes, obesity and insulin resistance. Inanother embodiment, the instructions of a package insert of an articleof manufacture or label of a kit comprise instructions foradministration of the antibody or fragment according to any of theaforementioned dose amounts, numbers of subsequent administrations, anddosing intervals between administrations, as well as any combination ofdose amounts numbers of subsequent administrations, and dosing intervalsbetween administrations described herein. In yet another embodiment, thecontainer of kit or article of manufacture is a pre-filled syringe.

It is to be understood that where the present specification mentionsmethods of treatments making use of antibodies or fragments thereof withcertain properties (such as Kd values or IC₅₀ values), this also meansto embody the use of such antibodies or fragments thereof in themanufacture of a medicament for use in these methods. Further, theinvention also encompasses antibodies or fragments thereof having theseproperties as well as pharmaceutical compositions comprising theseantibodies or fragments thereof for use in the methods of treatmentdiscussed hereinafter.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graph showing the results of an in vitro IL-1β inhibitionexperiment for the antibody designated AB7 and for Kineret® involvingIL-1 induced production of IL-8.

FIG. 2A is a graph showing the results of an in vivo IL-1β inhibitionexperiment for the antibodies designated AB5 and AB7 involving IL-1stimulated release of IL-6.

FIG. 2B is a graph showing the results of an in vivo IL-1β inhibitionexperiment for the antibodies designated AB7 involving IL-1 stimulatedrelease of IL-6, and comparing inhibition of human (panel A) versusmouse (panel B) IL-1β.

FIG. 3 is a graph showing serum concentrations following administration0.1, 1 or 10 mg/kg of an anti-IL-1β antibody in rats.

FIG. 4 is a graph showing serum concentrations following administrationof 0.3 or 3 mg/kg of an anti-IL-1β antibody in Cynomolgus monkeys.

FIG. 5 is a graph modeling plasma concentration profiles of ananti-IL-1β antibody in Cynomolgus monkeys following five monthly dosesof 0.1, 0.3, 1 or 3 mg/kg.

FIG. 6 is a table showing reduction of Staphyloccus epidermidis-inducedcytokine production in human whole blood by treatment with an anti-IL-1βantibody.

FIG. 7 is a graph showing the pharmacokinetics of AB7 in humansfollowing administration of a dose of 0.01 mg/kg of antibody.

FIG. 8 is a graph showing the effect on reducing CRP levels in humansfollowing administration of a 0.01 mg/kg dose of AB7.

FIG. 9 is a graph showing the effect on reducing CRP levels in humansfollowing administration of a 0.03 mg/kg dose of AB7.

FIG. 10 is a graph showing CRP levels in placebo controls from the 0.01and 0.03 mg/kg dose cohorts

FIG. 11 is a graph modeling the effect on CRP levels in humans followingadministration of various doses of an antibody with properties similarto AB7 at 28 day intervals.

FIG. 12 is graphs showing the effect of AB7 in a diet-induced obesitymouse model of Type 2 diabetes.

DETAILED DESCRIPTION

IL-1β is a pro-inflammatory cytokine secreted by a number of differentcell types including monocytes and macrophages. When released as part ofan inflammatory reaction, IL-1β produces a range of biological effects,mainly mediated through induction of other inflammatory mediators suchas corticotrophin, platelet factor-4, prostaglandin E2 (PGE2), IL-6, andIL-8. IL-1β induces both local and systemic inflammatory effects throughthe activation of the IL-1 receptor found on almost all cell types.

The interleukin-1 (IL-1) family of cytokines has been implicated inseveral disease states such as rheumatoid arthritis (RA),osteoarthritis, Crohn's disease, ulcerative colitis (UC), septic shock,chronic obstructive pulmonary disease (COPD), asthma, graft versus hostdisease, atherosclerosis, adult T-cell leukemia, multiple myeloma,multiple sclerosis, stroke, and Alzheimer's disease. IL-1 family membersinclude IL-1α, IL-1β, and IL-1Ra. Although related by their ability tobind to IL-1 receptors (IL-1R1, IL-1R2), each of these cytokines isexpressed by a different gene and has a different primary amino acidsequence. Furthermore, the physiological activities of these cytokinescan be distinguished from each other.

Compounds that disrupt IL-1 receptor signaling have been investigated astherapeutic agents to treat IL-1 mediated diseases, such as for examplesome of the aforementioned diseases. These compounds include recombinantIL-1Ra (Amgen Inc., Thousand Oaks, Calif.), IL-1 receptor “trap” peptide(Regeneron Inc., Tarrytown, N.Y.), as well as animal-derived IL-1βantibodies and recombinant IL-1β antibodies and fragments thereof.

As noted above, IL-1 receptor antagonist (IL-1Ra) polypeptide has beensuggested for use in the treatment of Type 2 diabetes (WO 2004/002512),but there remains a need for effective means to treat Type 2 diabetes,particularly those that do not require daily, repeated injections. Anadditional challenge for IL-1 receptor antagonist-based therapeutics isthe need for such therapeutics to occupy a large number of receptors,which is a formidable task since these receptors are widely expressed onall cells except red blood cells (Dinarello, Curr. Opin. Pharmacol.4:378-385, 2004). In most immune-mediated diseases, such as the diseasesdisclosed herein, the amount of IL-1β cytokine that is measurable inbody fluids or associated with activated cells is relatively low. Thus,a method of treatment and/or prevention that directly targets the IL-1βligand is a superior strategy, particularly when administering an IL-1βantibody with high affinity.

The present invention provides methods and related articles ofmanufacture for the treatment and/or prevention in mammals of Type 2diabetes, Type 1 diabetes, obesity, hyperglycemia, hyperinsulinemia,decreased insulin production, insulin resistance and/or disease statesand conditions characterized by insulin resistance, using an antibody orfragment thereof specific for IL-1β.

As shown in Example 1 below, we have surprisingly found that such anantibody (e.g., with high affinity) can be far more potent an inhibitorof the IL-1 pathway than is IL-Ra (e.g., Kineret®), and as shown inExample 9 below provides an opportunity to achieve a therapeutic effectat a lower dose and/or with less frequent administration than necessaryfor other drugs, such as recombinant IL-1Ra.

Such methods as described herein with an IL-1β antibody or fragment mayinclude the treatment of a subject suffering from Type 2 diabetes, Type1 diabetes, obesity, hyperglycemia, hyperinsulinemia, decreased insulinproduction, hypoinsulinemia, insulin resistance and/or disease statesand conditions characterized by insulin resistance. The methods also mayinclude preventing the occurrence of Type 2 diabetes, Type 1 diabetes,obesity, hyperglycemia, hyperinsulinemia, decreased insulin production,insulin resistance and disease states and conditions characterized byinsulin resistance or to prevent occurrence of the same in an at risksubject. As used herein, hyperinsulinemia refers to relativehyperinsulinemia due to insulin resistance.

Antibodies, Humanized Antibodies, and Human Engineered Antibodies

The IL-1 (e.g., IL-1β) binding antibodies of the present invention maybe provided as polyclonal antibodies, monoclonal antibodies (mAbs),recombinant antibodies, chimeric antibodies, CDR-grafted antibodies,fully human antibodies, single chain antibodies, and/or bispecificantibodies, as well as fragments, including variants and derivativesthereof, provided by known techniques, including, but not limited toenzymatic cleavage, peptide synthesis or recombinant techniques.

Antibodies generally comprise two heavy chain polypeptides and two lightchain polypeptides, though single domain antibodies having one heavychain and one light chain, and heavy chain antibodies devoid of lightchains are also contemplated. There are five types of heavy chains,called alpha, delta, epsilon, gamma and mu, based on the amino acidsequence of the heavy chain constant domain. These different types ofheavy chains give rise to five classes of antibodies, IgA (includingIgA₁ and IgA₂), IgD, IgE, IgG and IgM, respectively, including foursubclasses of IgG, namely IgG₁, IgG₂, IgG₃ and IgG₄. There are also twotypes of light chains, called kappa (κ) or lambda (λ) based on the aminoacid sequence of the constant domains. A full-length antibody includes aconstant domain and a variable domain. The constant region need not bepresent in an antigen binding fragment of an antibody. Antigen bindingfragments of an antibody disclosed herein can include Fab, Fab′,F(ab′)₂, and F(v) antibody fragments. As discussed in more detail below,IL-1β binding fragments encompass antibody fragments and antigen-bindingpolypeptides that will bind IL-1β.

Each of the heavy chain and light chain sequences of an antibody, orantigen binding fragment thereof, includes a variable region with threecomplementarity determining regions (CDRs) as well as non-CDR frameworkregions (FRs). The terms “heavy chain” and “light chain,” as usedherein, mean the heavy chain variable region and the light chainvariable region, respectively, unless otherwise noted. Heavy chain CDRsare referred to herein as CDR-H1, CDR-H2, and CDR-H3. Light chain CDRsare referred to herein as CDR-L1, CDR-L2, and CDR-L3. Variable regionsand CDRs in an antibody sequence can be identified (i) according togeneral rules that have been developed in the art or (ii) by aligningthe sequences against a database of known variable regions. Methods foridentifying these regions are described in Kontermann and Dubel, eds.,Antibody Engineering, Springer, New York, N.Y., 2001, and Dinarello etal., Current Protocols in Immunology, John Wiley and Sons Inc., Hoboken,N.J., 2000. Databases of antibody sequences are described in and can beaccessed through “The Kabatman” database at www.bioinf.org.uk/abs(maintained by A. C. Martin in the Department of Biochemistry &Molecular Biology University College London, London, England) and VBASE2at www.vbase2.org, as described in Retter et al., Nucl. Acids Res.,33(Database issue): D671-D674 (2005). The “Kabatman” database web sitealso includes general rules of thumb for identifying CDRs. The term“CDR,” as used herein, is as defined in Kabat et al., Sequences ofImmunological Interest, 5^(th) ed., U.S. Department of Health and HumanServices, 1991, unless otherwise indicated.

Polyclonal antibodies are preferably raised in animals by multiplesubcutaneous (sc) or intraperitoneal (ip) injections of the relevantantigen and an adjuvant. An improved antibody response may be obtainedby conjugating the relevant antigen to a protein that is immunogenic inthe species to be immunized, e.g., keyhole limpet hemocyanin, serumalbumin, bovine thyroglobulin, or soybean trypsin inhibitor using abifunctional or derivatizing agent, for example, maleimidobenzoylsulfosuccinimide ester (conjugation through cysteine residues),N-hydroxysuccinimide (through lysine residues), glutaraldehyde, succinicanhydride or other agents known in the art.

Animals are immunized against the antigen, immunogenic conjugates, orderivatives by combining, e.g., 100 μg or 5 μg of the protein orconjugate (for rabbits or mice, respectively) with 3 volumes of Freund'scomplete adjuvant and injecting the solution intradermally at multiplesites. One month later, the animals are boosted with ⅕ to 1/10 theoriginal amount of peptide or conjugate in Freund's complete adjuvant bysubcutaneous injection at multiple sites. At 7-14 days post-boosterinjection, the animals are bled and the serum is assayed for antibodytiter. Animals are boosted until the titer plateaus. Preferably, theanimal is boosted with the conjugate of the same antigen, but conjugatedto a different protein and/or through a different cross-linking reagent.Conjugates also can be made in recombinant cell culture as proteinfusions. Also, aggregating agents such as alum are suitably used toenhance the immune response.

Monoclonal antibody refers to an antibody obtained from a population ofsubstantially homogeneous antibodies. Monoclonal antibodies aregenerally highly specific, and may be directed against a singleantigenic site, in contrast to conventional (polyclonal) antibodypreparations that typically include different antibodies directedagainst different determinants (epitopes). In addition to theirspecificity, the monoclonal antibodies are advantageous in that they aresynthesized by the homogeneous culture, uncontaminated by otherimmunoglobulins with different specificities and characteristics.

Monoclonal antibodies to be used in accordance with the presentinvention may be made by the hybridoma method first described by Kohleret al., (Nature, 256:495-7, 1975), or may be made by recombinant DNAmethods (see, e.g., U.S. Pat. No. 4,816,567). The monoclonal antibodiesmay also be isolated from phage antibody libraries using the techniquesdescribed in, for example, Clackson et al., (Nature 352:624-628, 1991)and Marks et al., (J. Mol. Biol. 222:581-597, 1991).

In the hybridoma method, a mouse or other appropriate host animal, suchas a hamster or macaque monkey, is immunized as herein described toelicit lymphocytes that produce or are capable of producing antibodiesthat will specifically bind to the protein used for immunization.Alternatively, lymphocytes may be immunized in vitro. Lymphocytes thenare fused with myeloma cells using a suitable fusing agent, such aspolyethylene glycol, to form a hybridoma cell (Goding, MonoclonalAntibodies: Principles and Practice, pp. 59-103 (Academic Press, 1986)).

The hybridoma cells thus prepared are seeded and grown in a suitableculture medium that preferably contains one or more substances thatinhibit the growth or survival of the unfused, parental myeloma cells.For example, if the parental myeloma cells lack the enzyme hypoxanthineguanine phosphoribosyl transferase (HGPRT or HPRT), the culture mediumfor the hybridomas typically will include hypoxanthine, aminopterin, andthymidine (HAT medium), which substances prevent the growth ofHGPRT-deficient cells.

Preferred myeloma cells are those that fuse efficiently, support stablehigh-level production of antibody by the selected antibody-producingcells, and are sensitive to a medium. Human myeloma and mouse-humanheteromyeloma cell lines also have been described for the production ofhuman monoclonal antibodies (Kozbor, J. Immunol., 133: 3001 (1984);Brodeur et al., Monoclonal Antibody Production Techniques andApplications, pp. 51-63 (Marcel Dekker, Inc., New York, 1987)).Exemplary murine myeloma lines include those derived from MOP-21 andM.C.-11 mouse tumors available from the Salk Institute Cell DistributionCenter, San Diego, Calif. USA, and SP-2 or X63-Ag8-653 cells availablefrom the American Type Culture Collection, Rockville, Md. USA.

Culture medium in which hybridoma cells are growing is assayed forproduction of monoclonal antibodies directed against the antigen.Preferably, the binding specificity of monoclonal antibodies produced byhybridoma cells is determined by immunoprecipitation or by an in vitrobinding assay, such as radioimmunoassay (RIA) or enzyme-linkedimmunoabsorbent assay (ELISA). The binding affinity of the monoclonalantibody can, for example, be determined by Scatchard analysis (Munsonet al., Anal. Biochem., 107:220 (1980)).

After hybridoma cells are identified that produce antibodies of thedesired specificity, affinity, and/or activity, the clones may besubcloned by limiting dilution procedures and grown by standard methods(Goding, Monoclonal Antibodies: Principles and Practice, pp. 59-103(Academic Press, 1986)). Suitable culture media for this purposeinclude, for example, DMEM or RPMI-1640 medium. In addition, thehybridoma cells may be grown in vivo as ascites tumors in an animal. Themonoclonal antibodies secreted by the subclones are suitably separatedfrom the culture medium, ascites fluid, or serum by conventionalimmunoglobulin purification procedures such as, for example, proteinA-Sepharose, hydroxylapatite chromatography, gel electrophoresis,dialysis, or affinity chromatography.

It is further contemplated that antibodies of the invention may be usedas smaller antigen binding fragments of the antibody well-known in theart and described herein.

The present invention encompasses IL-1 (e.g., IL-1β) binding antibodiesthat include two full length heavy chains and two full length lightchains. Alternatively, the IL-1β binding antibodies can be constructssuch as single chain antibodies or “mini” antibodies that retain bindingactivity to IL-1β. Such constructs can be prepared by methods known inthe art such as, for example, the PCR mediated cloning and assembly ofsingle chain antibodies for expression in E. coli (as described inAntibody Engineering, The practical approach series, J. McCafferty, H.R. Hoogenboom, and D. J. Chiswell, editors, Oxford University Press,1996). In this type of construct, the variable portions of the heavy andlight chains of an antibody molecule are PCR amplified from cDNA. Theresulting amplicons are then assembled, for example, in a second PCRstep, through a linker DNA that encodes a flexible protein linkercomposed of the amino acids Gly and Ser. This linker allows the variableheavy and light chain portions to fold in such a way that the antigenbinding pocket is regenerated and antigen is bound with affinities oftencomparable to the parent full-length dimeric immunoglobulin molecule.

The IL-1 (e.g., IL-1β) binding antibodies and fragments of the presentinvention encompass variants of the exemplary antibodies, fragments andsequences disclosed herein. Variants include peptides and polypeptidescomprising one or more amino acid sequence substitutions, deletions,and/or additions that have the same or substantially the same affinityand specificity of epitope binding as one or more of the exemplaryantibodies, fragments and sequences disclosed herein. Thus, variantsinclude peptides and polypeptides comprising one or more amino acidsequence substitutions, deletions, and/or additions to the exemplaryantibodies, fragments and sequences disclosed herein where suchsubstitutions, deletions and/or additions do not cause substantialchanges in affinity and specificity of epitope binding. For example, avariant of an antibody or fragment may result from one or more changesto an antibody or fragment, where the changed antibody or fragment hasthe same or substantially the same affinity and specificity of epitopebinding as the starting sequence. Variants may be naturally occurring,such as allelic or splice variants, or may be artificially constructed.Variants may be prepared from the corresponding nucleic acid moleculesencoding said variants. Variants of the present antibodies and IL-1βbinding fragments may have changes in light and/or heavy chain aminoacid sequences that are naturally occurring or are introduced by invitro engineering of native sequences using recombinant DNA techniques.Naturally occurring variants include “somatic” variants which aregenerated in vivo in the corresponding germ line nucleotide sequencesduring the generation of an antibody response to a foreign antigen.

Variants of IL-1 (e.g., IL-1β) binding antibodies and binding fragmentsmay also be prepared by mutagenesis techniques. For example, amino acidchanges may be introduced at random throughout an antibody coding regionand the resulting variants may be screened for binding affinity forIL-1β or for another property. Alternatively, amino acid changes may beintroduced in selected regions of an IL-1β antibody, such as in thelight and/or heavy chain CDRs, and/or in the framework regions, and theresulting antibodies may be screened for binding to IL-1β or some otheractivity. Amino acid changes encompass one or more amino acidsubstitutions in a CDR, ranging from a single amino acid difference tothe introduction of multiple permutations of amino acids within a givenCDR, such as CDR3. In another method, the contribution of each residuewithin a CDR to IL-1β binding may be assessed by substituting at leastone residue within the CDR with alanine. Lewis et al. (1995), Mol.Immunol. 32: 1065-72. Residues which are not optimal for binding toIL-1β may then be changed in order to determine a more optimum sequence.Also encompassed are variants generated by insertion of amino acids toincrease the size of a CDR, such as CDR3. For example, most light chainCDR3 sequences are nine amino acids in length. Light chain sequences inan antibody which are shorter than nine residues may be optimized forbinding to IL-1 β by insertion of appropriate amino acids to increasethe length of the CDR.

Variants may also be prepared by “chain shuffling” of light or heavychains. Marks et al. (1992), Biotechnology 10: 779-83. A single light(or heavy) chain can be combined with a library having a repertoire ofheavy (or light) chains and the resulting population is screened for adesired activity, such as binding to IL-1β. This permits screening of agreater sample of different heavy (or light) chains in combination witha single light (or heavy) chain than is possible with librariescomprising repertoires of both heavy and light chains.

The IL-1 (e.g., IL-1β) binding antibodies and fragments of the presentinvention encompass derivatives of the exemplary antibodies, fragmentsand sequences disclosed herein. Derivatives include polypeptides orpeptides, or variants, fragments or derivatives thereof, which have beenchemically modified. Examples include covalent attachment of one or morepolymers, such as water soluble polymers, N-linked, or O-linkedcarbohydrates, sugars, phosphates, and/or other such molecules. Thederivatives are modified in a manner that is different from naturallyoccurring or starting peptide or polypeptides, either in the type orlocation of the molecules attached. Derivatives further include deletionof one or more chemical groups which are naturally present on thepeptide or polypeptide.

The IL-1β binding antibodies and fragments of the present invention canbe bispecific. Bispecific antibodies or fragments can be of severalconfigurations. For example, bispecific antibodies may resemble singleantibodies (or antibody fragments) but have two different antigenbinding sites (variable regions). Bispecific antibodies can be producedby chemical techniques (Kranz et al. (1981), Proc. Natl. Acad. Sci. USA,78: 5807), by “polydoma” techniques (U.S. Pat. No. 4,474,893) or byrecombinant DNA techniques. Bispecific antibodies of the presentinvention can have binding specificities for at least two differentepitopes, at least one of which is an epitope of IL-1β. The IL-1βbinding antibodies and fragments can also be heteroantibodies.Heteroantibodies are two or more antibodies, or antibody bindingfragments (Fab) linked together, each antibody or fragment having adifferent specificity.

Techniques for creating recombinant DNA versions of the antigen-bindingregions of antibody molecules which bypass the generation of monoclonalantibodies are contemplated for the present IL-1 (e.g., IL-1β) bindingantibodies and fragments. DNA is cloned into a bacterial expressionsystem. One example of such a technique suitable for the practice ofthis invention uses a bacteriophage lambda vector system having a leadersequence that causes the expressed Fab protein to migrate to theperiplasmic space (between the bacterial cell membrane and the cellwall) or to be secreted. One can rapidly generate and screen greatnumbers of functional Fab fragments for those which bind IL-1β. SuchIL-1β binding agents (Fab fragments with specificity for an IL-1βpolypeptide) are specifically encompassed within the IL-1β bindingantibodies and fragments of the present invention.

The present IL-1 (e.g., IL-1β) binding antibodies and fragments can behumanized or human engineered antibodies. As used herein, a humanizedantibody, or antigen binding fragment thereof, is a recombinantpolypeptide that comprises a portion of an antigen binding site from anon-human antibody and a portion of the framework and/or constantregions of a human antibody. A human engineered antibody or antibodyfragment is a non-human (e.g., mouse) antibody that has been engineeredby modifying (e.g., deleting, inserting, or substituting) amino acids atspecific positions so as to reduce or eliminate any detectableimmunogenicity of the modified antibody in a human.

Humanized antibodies include chimeric antibodies and CDR-graftedantibodies. Chimeric antibodies are antibodies that include a non-humanantibody variable region linked to a human constant region. Thus, inchimeric antibodies, the variable region is mostly non-human, and theconstant region is human. Chimeric antibodies and methods for makingthem are described in Morrison, et al., Proc. Natl. Acad. Sci. USA, 81:6841-6855 (1984), Boulianne, et al., Nature, 312: 643-646 (1984), andPCT Application Publication WO 86/01533. Although, they can be lessimmunogenic than a mouse monoclonal antibody, administrations ofchimeric antibodies have been associated with human anti-mouse antibodyresponses (HAMA) to the non-human portion of the antibodies. Chimericantibodies can also be produced by splicing the genes from a mouseantibody molecule of appropriate antigen-binding specificity togetherwith genes from a human antibody molecule of appropriate biologicalactivity, such as the ability to activate human complement and mediateADCC. Morrison et al. (1984), Proc. Natl. Acad. Sci., 81: 6851;Neuberger et al. (1984), Nature, 312: 604. One example is thereplacement of a Fc region with that of a different isotype.

CDR-grafted antibodies are antibodies that include the CDRs from anon-human “donor” antibody linked to the framework region from a human“recipient” antibody. Generally, CDR-grafted antibodies include morehuman antibody sequences than chimeric antibodies because they includeboth constant region sequences and variable region (framework) sequencesfrom human antibodies. Thus, for example, a CDR-grafted humanizedantibody of the invention can comprise a heavy chain that comprises acontiguous amino acid sequence (e.g., about 5 or more, 10 or more, oreven 15 or more contiguous amino acid residues) from the frameworkregion of a human antibody (e.g., FR-1, FR-2, or FR-3 of a humanantibody) or, optionally, most or all of the entire framework region ofa human antibody. CDR-grafted antibodies and methods for making them aredescribed in, Jones et al., Nature, 321: 522-525 (1986), Riechmann etal., Nature, 332: 323-327 (1988), and Verhoeyen et al., Science, 239:1534-1536 (1988)). Methods that can be used to produce humanizedantibodies also are described in U.S. Pat. Nos. 4,816,567, 5,721,367,5,837,243, and 6,180,377. CDR-grafted antibodies are considered lesslikely than chimeric antibodies to induce an immune reaction againstnon-human antibody portions. However, it has been reported thatframework sequences from the donor antibodies are required for thebinding affinity and/or specificity of the donor antibody, presumablybecause these framework sequences affect the folding of theantigen-binding portion of the donor antibody. Therefore, when donor,non-human CDR sequences are grafted onto unaltered human frameworksequences, the resulting CDR-grafted antibody can exhibit, in somecases, loss of binding avidity relative to the original non-human donorantibody. See, e.g., Riechmann et al., Nature, 332: 323-327 (1988), andVerhoeyen et al., Science, 239: 1534-1536 (1988).

Human engineered antibodies include for example “veneered” antibodiesand antibodies prepared using HUMAN ENGINEERING™ technology (U.S. Pat.No. 5,869,619). HUMAN ENGINEERING™ technology is commercially available,and involves altering an non-human antibody or antibody fragment, suchas a mouse or chimeric antibody or antibody fragment, by making specificchanges to the amino acid sequence of the antibody so as to produce amodified antibody with reduced immunogenicity in a human thatnonetheless retains the desirable binding properties of the originalnon-human antibodies. Generally, the technique involves classifyingamino acid residues of a non-human (e.g., mouse) antibody as “low risk”,“moderate risk”, or “high risk” residues. The classification isperformed using a global risk/reward calculation that evaluates thepredicted benefits of making particular substitution (e.g., forimmunogenicity in humans) against the risk that the substitution willaffect the resulting antibody's folding and/or antigen-bindingproperties. Thus, a low risk position is one for which a substitution ispredicted to be beneficial because it is predicted to reduceimmunogenicity without significantly affecting antigen bindingproperties. A moderate risk position is one for which a substitution ispredicted to reduce immunogenicity, but is more likely to affect proteinfolding and/or antigen binding. High risk positions contain residuesmost likely to be involved in proper folding or antigen binding.Generally, low risk positions in a non-human antibody are substitutedwith human residues, high risk positions are rarely substituted, andhumanizing substitutions at moderate risk positions are sometimes made,although not indiscriminately. Positions with prolines in the non-humanantibody variable region sequence are usually classified as at leastmoderate risk positions.

The particular human amino acid residue to be substituted at a given lowor moderate risk position of a non-human (e.g., mouse) antibody sequencecan be selected by aligning an amino acid sequence from the non-humanantibody's variable regions with the corresponding region of a specificor consensus human antibody sequence. The amino acid residues at low ormoderate risk positions in the non-human sequence can be substituted forthe corresponding residues in the human antibody sequence according tothe alignment. Techniques for making human engineered proteins aredescribed in greater detail in Studnicka et al., Protein Engineering, 7:805-814 (1994), U.S. Pat. Nos. 5,766,886, 5,770,196, 5,821,123, and5,869,619, and PCT Application Publication WO 93/11794.

“Veneered” antibodies are non-human or humanized (e.g., chimeric orCDR-grafted antibodies) antibodies that have been engineered to replacecertain solvent-exposed amino acid residues so as to further reducetheir immunogenicity or enhance their function. As surface residues of achimeric antibody are presumed to be less likely to affect properantibody folding and more likely to elicit an immune reaction, veneeringof a chimeric antibody can include, for instance, identifyingsolvent-exposed residues in the non-human framework region of a chimericantibody and replacing at least one of them with the correspondingsurface residues from a human framework region. Veneering can beaccomplished by any suitable engineering technique, including the use ofthe above-described HUMAN ENGINEERING™ technology.

In a different approach, a recovery of binding avidity can be achievedby “de-humanizing” a CDR-grafted antibody. De-humanizing can includerestoring residues from the donor antibody's framework regions to theCDR grafted antibody, thereby restoring proper folding. Similar“de-humanization” can be achieved by (i) including portions of the“donor” framework region in the “recipient” antibody or (ii) graftingportions of the “donor” antibody framework region into the recipientantibody (along with the grafted donor CDRs).

For a further discussion of antibodies, humanized antibodies, humanengineered, and methods for their preparation, see Kontermann and Dubel,eds., Antibody Engineering, Springer, New York, N.Y., 2001.

Exemplary humanized or human engineered antibodies include IgG, IgM,IgE, IgA, and IgD antibodies. The present antibodies can be of any class(IgG, IgA, IgM, IgE, IgD, etc.) or isotype and can comprise a kappa orlambda light chain. For example, a human antibody can comprise an IgGheavy chain or defined fragment, such as at least one of isotypes, IgG1,IgG2, IgG3 or IgG4. As a further example, the present antibodies orfragments can comprise an IgG1 heavy chain and an IgG1 light chain.

The present antibodies and fragments can be human antibodies, such asantibodies which bind IL-1β polypeptides and are encoded by nucleic acidsequences which are naturally occurring somatic variants of humangermline immunoglobulin nucleic acid sequence, and fragments, syntheticvariants, derivatives and fusions thereof. Such antibodies may beproduced by any method known in the art, such as through the use oftransgenic mammals (such as transgenic mice) in which the nativeimmunoglobulin repertoire has been replaced with human V-genes in themammal chromosome. Such mammals appear to carry out VDJ recombinationand somatic hypermutation of the human germline antibody genes in anormal fashion, thus producing high affinity antibodies with completelyhuman sequences.

Human antibodies to target protein can also be produced using transgenicanimals that have no endogenous immunoglobulin production and areengineered to contain human immunoglobulin loci. For example, WO98/24893 discloses transgenic animals having a human Ig locus whereinthe animals do not produce functional endogenous immunoglobulins due tothe inactivation of endogenous heavy and light chain loci. WO 91/00906also discloses transgenic non-primate mammalian hosts capable ofmounting an immune response to an immunogen, wherein the antibodies haveprimate constant and/or variable regions, and wherein the endogenousimmunoglobulin encoding loci are substituted or inactivated. WO 96/30498and U.S. Pat. No. 6,091,001 disclose the use of the Cre/Lox system tomodify the immunoglobulin locus in a mammal, such as to replace all or aportion of the constant or variable region to form a modified antibodymolecule. WO 94/02602 discloses non-human mammalian hosts havinginactivated endogenous Ig loci and functional human Ig loci. U.S. Pat.No. 5,939,598 discloses methods of making transgenic mice in which themice lack endogenous heavy chains, and express an exogenousimmunoglobulin locus comprising one or more xenogeneic constant regions.See also, U.S. Pat. Nos. 6,114,598 6,657,103 and 6,833,268.

Using a transgenic animal described above, an immune response can beproduced to a selected antigenic molecule, and antibody producing cellscan be removed from the animal and used to produce hybridomas thatsecrete human monoclonal antibodies. Immunization protocols, adjuvants,and the like are known in the art, and are used in immunization of, forexample, a transgenic mouse as described in WO 96/33735. Thispublication discloses monoclonal antibodies against a variety ofantigenic molecules including IL-6, IL-8, TNFa, human CD4, L selectin,gp39, and tetanus toxin. The monoclonal antibodies can be tested for theability to inhibit or neutralize the biological activity orphysiological effect of the corresponding protein. WO 96/33735 disclosesthat monoclonal antibodies against IL-8, derived from immune cells oftransgenic mice immunized with IL-8, blocked IL-8 induced functions ofneutrophils. Human monoclonal antibodies with specificity for theantigen used to immunize transgenic animals are also disclosed in WO96/34096 and U.S. patent application no. 20030194404; and U.S. patentapplication no. 20030031667.

Additional transgenic animals useful to make monoclonal antibodiesinclude the Medarex HuMAb-MOUSE®, described in U.S. Pat. No. 5,770,429and Fishwild, et al. (Nat. Biotechnol. 14:845-851, 1996), which containsgene sequences from unrearranged human antibody genes that code for theheavy and light chains of human antibodies. Immunization of aHuMAb-MOUSE® enables the production of fully human monoclonal antibodiesto the target protein.

Also, Ishida et al. (Cloning Stem Cells. 4:91-102, 2002) describes theTransChromo Mouse (TCMOUSE™) which comprises megabase-sized segments ofhuman DNA and which incorporates the entire human immunoglobulin (Mg)loci. The TCMOUSE™ has a fully diverse repertoire of hIgs, including allthe subclasses of IgGs (IgG1-G4). Immunization of the TC MOUSE™ withvarious human antigens produces antibody responses comprising humanantibodies.

See also Jakobovits et al., Proc. Natl. Acad. Sci. USA, 90:2551 (1993);Jakobovits et al., Nature, 362:255-258 (1993); Bruggermann et al., Yearin Immunol., 7:33 (1993); and U.S. Pat. No. 5,591,669, U.S. Pat. No.5,589,369, U.S. Pat. No. 5,545,807; and U.S. Patent Publication No.20020199213. U.S. Patent Publication No. 20030092125 describes methodsfor biasing the immune response of an animal to the desired epitope.Human antibodies may also be generated by in vitro activated B cells(see U.S. Pat. Nos. 5,567,610 and 5,229,275).

Human antibodies can also be generated through the in vitro screening ofantibody display libraries. See Hoogenboom et al. (1991), J. Mol. Biol.227: 381; and Marks et al. (1991), J. Mol. Biol. 222: 581. Variousantibody-containing phage display libraries have been described and maybe readily prepared. Libraries may contain a diversity of human antibodysequences, such as human Fab, Fv, and scFv fragments, that may bescreened against an appropriate target. Phage display libraries maycomprise peptides or proteins other than antibodies which may bescreened to identify selective binding agents of IL-1β.

The development of technologies for making repertoires of recombinanthuman antibody genes, and the display of the encoded antibody fragmentson the surface of filamentous bacteriophage, has provided a means formaking human antibodies directly. The antibodies produced by phagetechnology are produced as antigen binding fragments-usually Fv or Fabfragments-in bacteria and thus lack effector functions. Effectorfunctions can be introduced by one of two strategies: The fragments canbe engineered either into complete antibodies for expression inmammalian cells, or into bispecific antibody fragments with a secondbinding site capable of triggering an effector function.

The invention contemplates a method for producing target-specificantibody or antigen-binding portion thereof comprising the steps ofsynthesizing a library of human antibodies on phage, screening thelibrary with target protein or a portion thereof, isolating phage thatbind target, and obtaining the antibody from the phage. By way ofexample, one method for preparing the library of antibodies for use inphage display techniques comprises the steps of immunizing a non-humananimal comprising human immunoglobulin loci with target antigen or anantigenic portion thereof to create an immune response, extractingantibody producing cells from the immunized animal; isolating RNA fromthe extracted cells, reverse transcribing the RNA to produce cDNA,amplifying the cDNA using a primer, and inserting the cDNA into a phagedisplay vector such that antibodies are expressed on the phage.Recombinant target-specific antibodies of the invention may be obtainedin this way.

Phage-display processes mimic immune selection through the display ofantibody repertoires on the surface of filamentous bacteriophage, andsubsequent selection of phage by their binding to an antigen of choice.One such technique is described in WO 99/10494, which describes theisolation of high affinity and functional agonistic antibodies for MPLand msk receptors using such an approach. Antibodies of the inventioncan be isolated by screening of a recombinant combinatorial antibodylibrary, preferably a scFv phage display library, prepared using humanV_(L) and V_(H) cDNAs prepared from mRNA derived from human lymphocytes.Methodologies for preparing and screening such libraries are known inthe art. See e.g., U.S. Pat. No. 5,969,108. There are commerciallyavailable kits for generating phage display libraries (e.g., thePharmacia Recombinant Phage Antibody System, catalog no. 27-9400-01; andthe Stratagene SurfZAP™ phage display kit, catalog no. 240612). Thereare also other methods and reagents that can be used in generating andscreening antibody display libraries (see, e.g., Ladner et al. U.S. Pat.No. 5,223,409; Kang et al. PCT Publication No. WO 92/18619; Dower et al.PCT Publication No. WO 91/17271; Winter et al. PCT Publication No. WO92/20791; Markland et al. PCT Publication No. WO 92/15679; Breitling etal. PCT Publication No. WO 93/01288; McCafferty et al. PCT PublicationNo. WO 92/01047; Garrard et al. PCT Publication No. WO 92/09690; Fuchset al. (1991) Bio/Technology 9:1370-1372; Hay et al. (1992) Hum.Antibod. Hybridomas 3:81-85; Huse et al. (1989) Science 246:1275-1281;McCafferty et al., Nature (1990) 348:552-554; Griffiths et al. (1993)EMBO J. 12:725-734; Hawkins et al. (1992) J. Mol. Biol. 226:889-896;Clackson et al. (1991) Nature 352:624-628; Gram et al. (1992) Proc.Natl. Acad. Sci. USA 89:3576-3580; Garrad et al. (1991) Bio/Technology9:1373-1377; Hoogenboom et al. (1991) Nuc Acid Res 19:4133-4137; andBarbas et al. (1991) Proc. Natl. Acad. Sci. USA 88:7978-7982.

In one embodiment, to isolate human antibodies specific for the targetantigen with the desired characteristics, a human V_(H) and V_(L)library are screened to select for antibody fragments having the desiredspecificity. The antibody libraries used in this method are preferablyscFv libraries prepared and screened as described herein and in the art(McCafferty et al., PCT Publication No. WO 92/01047, McCafferty et al.,(Nature 348:552-554, 1990); and Griffiths et al., (EMBO J. 12:725-734,1993). The scFv antibody libraries preferably are screened using targetprotein as the antigen.

Alternatively, the Fd fragment (V_(H)-C_(H)1) and light chain(V_(L)-C_(L)) of antibodies are separately cloned by PCR and recombinedrandomly in combinatorial phage display libraries, which can then beselected for binding to a particular antigen. The Fab fragments areexpressed on the phage surface, i.e., physically linked to the genesthat encode them. Thus, selection of Fab by antigen binding co-selectsfor the Fab encoding sequences, which can be amplified subsequently.Through several rounds of antigen binding and re-amplification, aprocedure termed panning, Fab specific for the antigen are enriched andfinally isolated.

In 1994, an approach for the humanization of antibodies, called “guidedselection”, was described. Guided selection utilizes the power of thephage display technique for the humanization of mouse monoclonalantibody (See Jespers, L. S., et al., Bio/Technology 12, 899-903(1994)). For this, the Fd fragment of the mouse monoclonal antibody canbe displayed in combination with a human light chain library, and theresulting hybrid Fab library may then be selected with antigen. Themouse Fd fragment thereby provides a template to guide the selection.Subsequently, the selected human light chains are combined with a humanFd fragment library. Selection of the resulting library yields entirelyhuman Fab.

A variety of procedures have been described for deriving humanantibodies from phage-display libraries (See, for example, Hoogenboom etal., J. Mol. Biol., 227:381 (1991); Marks et al., J. Mol. Biol,222:581-597 (1991); U.S. Pat. Nos. 5,565,332 and 5,573,905; Clackson,T., and Wells, J. A., TIBTECH 12, 173-184 (1994)). In particular, invitro selection and evolution of antibodies derived from phage displaylibraries has become a powerful tool (See Burton, D. R., and Barbas III,C. F., Adv. Immunol. 57, 191-280 (1994); Winter, G., et al., Annu. Rev.Immunol. 12, 433-455 (1994); U.S. patent publication no. 20020004215 andWO 92/01047; U.S. patent publication no. 20030190317; and U.S. Pat. Nos.6,054,287 and 5,877,293.

Watkins, “Screening of Phage-Expressed Antibody Libraries by CaptureLift,” Methods in Molecular Biology, Antibody Phage Display: Methods andProtocols 178: 187-193 (2002), and U.S. patent publication no.20030044772, published Mar. 6, 2003, describe methods for screeningphage-expressed antibody libraries or other binding molecules by capturelift, a method involving immobilization of the candidate bindingmolecules on a solid support.

Fv fragments are displayed on the surface of phage, by the associationof one chain expressed as a phage protein fusion (e.g., with M13 geneIII) with the complementary chain expressed as a soluble fragment. It iscontemplated that the phage may be a filamentous phage such as one ofthe class I phages: fd, M13, f1, If1, Ike, ZJ/Z, Ff and one of the classII phages Xf, Pf1 and Pf3. The phage may be M13, or fd or a derivativethereof.

Once initial human V_(L) and V_(H) segments are selected, “mix andmatch” experiments, in which different pairs of the initially selectedV_(L) and V_(H) segments are screened for target binding, are performedto select preferred V_(L)/V_(H) pair combinations. Additionally, tofurther improve the quality of the antibody, the V_(L) and V_(H)segments of the preferred V_(L)/V_(H) pair(s) can be randomly mutated,preferably within the any of the CDR1, CDR2 or CDR3 region of V_(H)and/or V_(L), in a process analogous to the in vivo somatic mutationprocess responsible for affinity maturation of antibodies during anatural immune response. This in vitro affinity maturation can beaccomplished by amplifying V_(L) and V_(H) regions using PCR primerscomplimentary to the V_(H) CDR1, CDR2, and CDR3, or V_(L) CDR1, CDR2,and CDR3, respectively, which primers have been “spiked” with a randommixture of the four nucleotide bases at certain positions such that theresultant PCR products encode V_(L) and V_(H) segments into which randommutations have been introduced into the V_(H) and/or V_(L) CDR3 regions.These randomly mutated V_(L) and V_(H) segments can be rescreened forbinding to target antigen.

Following screening and isolation of an target specific antibody from arecombinant immunoglobulin display library, nucleic acid encoding theselected antibody can be recovered from the display package (e.g., fromthe phage genome) and subcloned into other expression vectors bystandard recombinant DNA techniques. If desired, the nucleic acid can befurther manipulated to create other antibody forms of the invention, asdescribed below. To express a recombinant human antibody isolated byscreening of a combinatorial library, the DNA encoding the antibody iscloned into a recombinant expression vector and introduced into amammalian host cell, as described herein.

It is contemplated that the phage display method may be carried out in amutator strain of bacteria or host cell. A mutator strain is a host cellwhich has a genetic defect which causes DNA replicated within it to bemutated with respect to its parent DNA. Example mutator strains areNR9046mutD5 and NR9046 mut T1.

It is also contemplated that the phage display method may be carried outusing a helper phage. This is a phage which is used to infect cellscontaining a defective phage genome and which functions to complementthe defect. The defective phage genome can be a phagemid or a phage withsome function encoding gene sequences removed. Examples of helper phagesare M13K07, M13K07 gene III no. 3; and phage displaying or encoding abinding molecule fused to a capsid protein.

Antibodies are also generated via phage display screening methods usingthe hierarchical dual combinatorial approach as disclosed in WO 92/01047in which an individual colony containing either an H or L chain clone isused to infect a complete library of clones encoding the other chain (Lor H) and the resulting two-chain specific binding member is selected inaccordance with phage display techniques such as those describedtherein. This technique is also disclosed in Marks et al,(Bio/Technology, 10:779-783, 1992).

Methods for display of peptides on the surface of yeast and microbialcells have also been used to identify antigen specific antibodies. See,for example, U.S. Pat. No. 6,699,658. Antibody libraries may be attachedto yeast proteins, such as agglutinin, effectively mimicking the cellsurface display of antibodies by B cells in the immune system.

In addition to phage display methods, antibodies may be isolated usingribosome mRNA display methods and microbial cell display methods.Selection of polypeptide using ribosome display is described in Hanes etal., (Proc. Natl Acad Sci USA, 94:4937-4942, 1997) and U.S. Pat. Nos.5,643,768 and 5,658,754 issued to Kawasaki. Ribosome display is alsouseful for rapid large scale mutational analysis of antibodies. Theselective mutagenesis approach also provides a method of producingantibodies with improved activities that can be selected using ribosomaldisplay techniques.

The IL-1 (e.g., IL-1β) binding antibodies and fragments may comprise oneor more portions that do not bind IL-1β but instead are responsible forother functions, such as circulating half-life, direct cytotoxic effect,detectable labeling, or activation of the recipient's endogenouscomplement cascade or endogenous cellular cytotoxicity. The antibodiesor fragments may comprise all or a portion of the constant region andmay be of any isotype, including IgA (e.g., IgA1 or IgA2), IgD, IgE, IgG(e.g. IgG1, IgG2, IgG3 or IgG4), or IgM. In addition to, or instead of,comprising a constant region, antigen-binding compounds of the inventionmay include an epitope tag, a salvage receptor epitope, a label moietyfor diagnostic or purification purposes, or a cytotoxic moiety such as aradionuclide or toxin.

The constant region (when present) of the present antibodies andfragments may be of the γ1, γ2, γ3, γ4, μ, β2, or δ or ε type,preferably of the γ type, more preferably of the y, type, whereas theconstant part of a human light chain may be of the κ or λ type (whichincludes the λ₁, λ₂ and λ₃ subtypes) but is preferably of the K type.

Variants also include antibodies or fragments comprising a modified Fcregion, wherein the modified Fc region comprises at least one amino acidmodification relative to a wild-type Fc region. The variant Fc regionmay be designed, relative to a comparable molecule comprising thewild-type Fc region, so as to bind Fc receptors with a greater or lesseraffinity.

For example, the present IL-1β binding antibodies and fragments maycomprise a modified Fc region. Fc region refers to naturally-occurringor synthetic polypeptides homologous to the IgG C-terminal domain thatis produced upon papain digestion of IgG. IgG Fc has a molecular weightof approximately 50 kD. In the present antibodies and fragments, anentire Fc region can be used, or only a half-life enhancing portion. Inaddition, many modifications in amino acid sequence are acceptable, asnative activity is not in all cases necessary or desired.

The Fc region can be mutated, if desired, to inhibit its ability to fixcomplement and bind the Fc receptor with high affinity. For murine IgGFc, substitution of Ala residues for Glu 318, Lys 320, and Lys 322renders the protein unable to direct ADCC. Substitution of Glu for Leu235 inhibits the ability of the protein to bind the Fc receptor withhigh affinity. Various mutations for human IgG also are known (see,e.g., Morrison et al., 1994, The Immunologist 2: 119 124 and Brekke etal., 1994, The Immunologist 2: 125).

In some embodiments, the present an antibodies or fragments are providedwith a modified Fc region where a naturally-occurring Fc region ismodified to increase the half-life of the antibody or fragment in abiological environment, for example, the serum half-life or a half-lifemeasured by an in vitro assay. Methods for altering the original form ofa Fc region of an IgG also are described in U.S. Pat. No. 6,998,253.

In certain embodiments, it may be desirable to modify the antibody orfragment in order to increase its serum half-life, for example, addingmolecules such as PEG or other water soluble polymers, includingpolysaccharide polymers, to antibody fragments to increase thehalf-life. This may also be achieved, for example, by incorporation of asalvage receptor binding epitope into the antibody fragment (e.g., bymutation of the appropriate region in the antibody fragment or byincorporating the epitope into a peptide tag that is then fused to theantibody fragment at either end or in the middle, e.g., by DNA orpeptide synthesis) (see, International Publication No. WO96/32478).Salvage receptor binding epitope refers to an epitope of the Fc regionof an IgG molecule (e.g., IgG₁, IgG₂, IgG₃, or IgG₄) that is responsiblefor increasing the in vivo serum half-life of the IgG molecule.

A salvage receptor binding epitope can include a region wherein any oneor more amino acid residues from one or two loops of a Fc domain aretransferred to an analogous position of the antibody fragment. Even morepreferably, three or more residues from one or two loops of the Fcdomain are transferred. Still more preferred, the epitope is taken fromthe CH2 domain of the Fc region (e.g., of an IgG) and transferred to theCH1, CH3, or V_(H) region, or more than one such region, of theantibody. Alternatively, the epitope is taken from the CH2 domain of theFc region and transferred to the C_(L) region or V_(L) region, or both,of the antibody fragment. See also International applications WO97/34631 and WO 96/32478 which describe Fc variants and theirinteraction with the salvage receptor.

Mutation of residues within Fc receptor binding sites can result inaltered effector function, such as altered ADCC or CDC activity, oraltered half-life. Potential mutations include insertion, deletion orsubstitution of one or more residues, including substitution withalanine, a conservative substitution, a non-conservative substitution,or replacement with a corresponding amino acid residue at the sameposition from a different IgG subclass (e.g. replacing an IgG1 residuewith a corresponding IgG2 residue at that position). For example it hasbeen reported that mutating the serine at amino acid position 241 inIgG4 to proline (found at that position in IgG1 and IgG2) led to theproduction of a homogeneous antibody, as well as extending serumhalf-life and improving tissue distribution compared to the originalchimeric IgG4. (Angal et al., Mol. Immunol. 30:105-8, 1993).

Antibody fragments are portions of an intact full length antibody, suchas an antigen binding or variable region of the intact antibody.Examples of antibody fragments include Fab, Fab′, F(ab′)₂, and Fvfragments; diabodies; linear antibodies; single-chain antibody molecules(e.g., scFv); multispecific antibody fragments such as bispecific,trispecific, and multispecific antibodies (e.g., diabodies, triabodies,tetrabodies); minibodies; chelating recombinant antibodies; tribodies orbibodies; intrabodies; nanobodies; small modular immunopharmaceuticals(SMIP), adnectins, binding-domain immunoglobulin fusion proteins;camelized antibodies; V_(HH) containing antibodies; and any otherpolypeptides formed from antibody fragments.

The present invention includes IL-1β binding antibody fragmentscomprising any of the foregoing heavy or light chain sequences and whichbind IL-1β. The term fragments as used herein refers to any 3 or morecontiguous amino acids (e.g., 4 or more, 5 or more 6 or more, 8 or more,or even 10 or more contiguous amino acids) of the antibody andencompasses Fab, Fab′, F(ab′)₂, and F(v) fragments, or the individuallight or heavy chain variable regions or portion thereof. IL-1β bindingfragments include, for example, Fab, Fab′, F(ab′)₂, Fv and scFv. Thesefragments lack the Fc fragment of an intact antibody, clear more rapidlyfrom the circulation, and can have less non-specific tissue binding thanan intact antibody. See Wahl et al. (1983), J. Nucl. Med., 24: 316-25.These fragments can be produced from intact antibodies using well knownmethods, for example by proteolytic cleavage with enzymes such as papain(to produce Fab fragments) or pepsin (to produce F(ab′)₂ fragments).

In vitro and cell based assays are well described in the art for use indetermining binding of IL-1β to IL-1 receptor type I (IL-1RI), includingassays that determining in the presence of molecules (such asantibodies, antagonists, or other inhibitors) that bind to IL-1β orIL-1RI. (see for example Evans et al., (1995), J. Biol. Chem.270:11477-11483; Vigers et al., (2000), J. Biol. Chem. 275:36927-36933;Yanofsky et al., (1996), Proc. Natl. Acad. Sci. USA 93:7381-7386;Fredericks et al., (2004), Protein Eng. Des. Sel. 17:95-106; Slack etal., (1993), J. Biol. Chem. 268:2513-2524; Smith et al., (2003),Immunity 18:87-96; Vigers et al., (1997), Nature 386:190-194; Ruggieroet al., (1997), J. Immunol. 158:3881-3887; Guo et al., (1995), J. Biol.Chem. 270:27562-27568; Svenson et al., (1995), Eur. J. Immunol.25:2842-2850; Arend et al., (1994), J. Immunol. 153:4766-4774).Recombinant IL-1 receptor type I, including human IL-1 receptor type I,for such assays is readily available from a variety of commercialsources (see for example R&D Systems, SIGMA). IL-1 receptor type I alsocan be expressed from an expression construct or vector introduced intoan appropriate host cell using standard molecular biology andtransfection techniques known in the art. The expressed IL-1 receptortype I may then be isolated and purified for use in binding assays, oralternatively used directly in a cell associated form.

For example, the binding of IL-1β to IL-1 receptor type I may bedetermined by immobilizing an IL-1β binding antibody, contacting IL-1βwith the immobilized antibody and determining whether the IL-1β wasbound to the antibody, and contacting a soluble form of IL-1RI with thebound IL-1β/antibody complex and determining whether the soluble IL-1RIwas bound to the complex. The protocol may also include contacting thesoluble IL-1RI with the immobilized antibody before the contact withIL-1β, to confirm that the soluble IL-1RI does not bind to theimmobilized antibody. This protocol can be performed using a Biacore®instrument for kinetic analysis of binding interactions. Such a protocolcan also be employed to determine whether an antibody or other moleculepermits or blocks the binding of IL-1β to IL-1 receptor type I.

For other IL-1β/IL-1RI binding assays, the permitting or blocking ofIL-1β binding to IL-1 receptor type I may be determined by comparing thebinding of IL-1β to IL-1RI in the presence or absence of IL-1βantibodies or IL-1β binding fragments thereof. Blocking is identified inthe assay readout as a designated reduction of IL-1β binding to IL-1receptor type I in the presence of anti-IL-1β antibodies or IL-1βbinding fragments thereof, as compared to a control sample that containsthe corresponding buffer or diluent but not an IL-1β antibody or IL-1βbinding fragment thereof. The assay readout may be qualitatively viewedas indicating the presence or absence of blocking, or may bequantitatively viewed as indicating a percent or fold reduction inbinding due to the presence of the antibody or fragment.

Alternatively or additionally, when an IL-1β binding antibody or IL-1βbinding fragment substantially blocks IL-1β binding to IL-1RI, the IL-1βbinding to IL-1RI is reduced by at least 10-fold, alternatively at leastabout 20-fold, alternatively at least about 50-fold, alternatively atleast about 100-fold, alternatively at least about 1000-fold,alternatively at least about 10000-fold, or more, compared to binding ofthe same concentrations of IL-1β and IL-1RI in the absence of theantibody or fragment. As another example, when an IL-1β binding antibodyor IL-1β binding fragment substantially permits IL-1β binding to IL-1RI,the IL-1β binding to IL-1RI is at least about 90%, alternatively atleast about 95%, alternatively at least about 99%, alternatively atleast about 99.9%, alternatively at least about 99.99%, alternatively atleast about 99.999%, alternatively at least about 99.9999%,alternatively substantially identical to binding of the sameconcentrations of IL-1β and IL-1RI in the absence of the antibody orfragment.

The present invention may in certain embodiments encompass IL-1β bindingantibodies or IL-1β binding fragments that bind to the same epitope orsubstantially the same epitope as one or more of the exemplaryantibodies described herein. Alternatively or additionally, the IL-1βbinding antibodies or IL-1β binding fragments compete with the bindingof an antibody having variable region sequences of AB7, described inU.S. application Ser. No. 11/472,813 (sequences shown below).Alternatively or additionally, the present invention encompasses IL-1βbinding antibodies and fragments that bind to an epitope contained inthe amino acid sequence ESVDPKNYPKKKMEKRFVFNKIE (SEQ ID NO: 1), anepitope that the antibodies designated AB5 and AB7 (U.S. applicationSer. No. 11/472,813) bind to. As contemplated herein, one can readilydetermine if an IL-1β binding antibody or fragment binds to the sameepitope or substantially the same epitope as one or more of theexemplary antibodies, such as for example the antibody designated AB7,using any of several known methods in the art.

For example, the key amino acid residues (epitope) bound by an IL-1βbinding antibody or fragment may be determined using a peptide array,such as for example, a PepSpot™ peptide array (JPT Peptide Technologies,Berlin, Germany), wherein a scan of twelve amino-acid peptides, spanningthe entire IL-1β amino acid sequence, each peptide overlapping by 11amino acid to the previous one, is synthesized directly on a membrane.The membrane carrying the peptides is then probed with the antibody forwhich epitope binding information is sought, for example at aconcentration of 2 μg/ml, for 2 hr at room temperature. Binding ofantibody to membrane bound peptides may be detected using a secondaryHRP-conjugated goat anti-human (or mouse, when appropriate) antibody,followed by enhanced chemiluminescence (ECL). The peptides spot(s)corresponding to particular amino acid residues or sequences of themature IL-1β protein, and which score positive for antibody binding, areindicative of the epitope bound by the particular antibody.

Alternatively or in addition, antibody competition experiments may beperformed and such assays are well known in the art. For example, todetermine if an antibody or fragment binds to an epitope contained in apeptide sequence comprising the amino acids ESVDPKNYPKKKMEKRFVFNKIE (SEQID NO: 1), which corresponds to residues 83-105 of the mature IL-1βprotein, an antibody of unknown specificity may be compared with any ofthe exemplary of antibodies (e.g., AB7) of the present invention thatare known to bind an epitope contained within this sequence. Bindingcompetition assays may be performed, for example, using a Biacore®instrument for kinetic analysis of binding interactions or by ELISA. Insuch an assay, the antibody of unknown epitope specificity is evaluatedfor its ability to compete for binding against the known comparatorantibody (e.g., AB7). Competition for binding to a particular epitope isdetermined by a reduction in binding to the IL-1β epitope of at leastabout 50%, or at least about 70%, or at least about 80%, or at leastabout 90%, or at least about 95%, or at least about 99% or about 100%for the known comparator antibody (e.g., AB7) and is indicative ofbinding to substantially the same epitope.

In view of the identification in this disclosure of IL-1β bindingregions in exemplary antibodies and/or epitopes recognized by thedisclosed antibodies, it is contemplated that additional antibodies withsimilar binding characteristics and therapeutic or diagnostic utilitycan be generated that parallel the embodiments of this disclosure.

Antigen-binding fragments of an antibody include fragments that retainthe ability to specifically bind to an antigen, generally by retainingthe antigen-binding portion of the antibody. It is well established thatthe antigen-binding function of an antibody can be performed byfragments of a full-length antibody. Examples of antigen-bindingportions include (i) a Fab fragment, which is a monovalent fragmentconsisting of the VL, VH, CL and CH1 domains; (ii) a F(ab′)² fragment,which is a bivalent fragment comprising two Fab fragments linked by adisulfide bridge at the hinge region; (iii) a Fd fragment which is theVH and CH1 domains; (iv) a Fv fragment which is the VL and VH domains ofa single arm of an antibody, (v) a dAb fragment (Ward et al., (1989)Nature 341:544-546), which is a VH domain; and (vi) an isolatedcomplementarity determining region (CDR). Single chain antibodies arealso encompassed within the term antigen-binding portion of an antibody.The IL-1β binding antibodies and fragments of the present invention alsoencompass monovalent or multivalent, or monomeric or multimeric (e.g.tetrameric), CDR-derived binding domains with or without a scaffold (forexample, protein or carbohydrate scaffolding).

The present IL-1β binding antibodies or fragments may be part of alarger immunoadhesion molecules, formed by covalent or non-covalentassociation of the antibody or antibody portion with one or more otherproteins or peptides. Examples of such immunoadhesion molecules includeuse of the streptavidin core region to make a tetrameric scFv molecule(Kipriyanov, S. M., et al. (1995) Human Antibodies and Hybridomas6:93-101) and use of a cysteine residue, a marker peptide and aC-terminal polyhistidine tag to make bivalent and biotinylated scFvmolecules (Kipriyanov, S. M., et al. (1994) Mol. Immunol. 31:1047-1058).Antibodies and fragments comprising immunoadhesion molecules can beobtained using standard recombinant DNA techniques, as described herein.Preferred antigen binding portions are complete domains or pairs ofcomplete domains.

The IL-1β binding antibodies and fragments of the present invention alsoencompass domain antibody (dAb) fragments (Ward et al., Nature341:544-546, 1989) which consist of a V_(H) domain. The IL-1β bindingantibodies and fragments of the present invention also encompassdiabodies, which are bivalent antibodies in which V_(H) and V_(L)domains are expressed on a single polypeptide chain, but using a linkerthat is too short to allow for pairing between the two domains on thesame chain, thereby forcing the domains to pair with complementarydomains of another chain and creating two antigen binding sites (seee.g., EP 404,097; WO 93/11161; Holliger et al., Proc. Natl. Acad. Sci.USA 90:6444-6448, 1993, and Poljak et al., Structure 2:1121-1123, 1994).Diabodies can be bispecific or monospecific.

The IL-1β binding antibodies and fragments of the present invention alsoencompass single-chain antibody fragments (scFv) that bind to IL-1β. AnscFv comprises an antibody heavy chain variable region (V_(H)) operablylinked to an antibody light chain variable region (V_(L)) wherein theheavy chain variable region and the light chain variable region,together or individually, form a binding site that binds IL-1β. An scFvmay comprise a V_(H) region at the amino-terminal end and a V_(L) regionat the carboxy-terminal end. Alternatively, scFv may comprise a V_(L)region at the amino-terminal end and a V_(H) region at thecarboxy-terminal end. Furthermore, although the two domains of the Fvfragment, V_(L) and V_(H), are coded for by separate genes, they can bejoined, using recombinant methods, by a synthetic linker that enablesthem to be made as a single protein chain in which the V_(L) and V_(H)regions pair to form monovalent molecules (known as single chain Fv(scFv); see e.g., Bird et al. (1988) Science 242:423-426; and Huston etal. (1988) Proc. Natl. Acad. Sci. USA 85:5879-5883).

An scFv may optionally further comprise a polypeptide linker between theheavy chain variable region and the light chain variable region. Suchpolypeptide linkers generally comprise between 1 and 50 amino acids,alternatively between 3 and 12 amino acids, alternatively 2 amino acids.An example of a linker peptide for linking heavy and light chains in anscFv comprises the 5 amino acid sequence Gly-Gly-Gly-Gly-Ser (SEQ ID NO:2). Other examples comprise one or more tandem repeats of this sequence(for example, a polypeptide comprising two to four repeats ofGly-Gly-Gly-Gly-Ser (SEQ ID NO: 2) to create linkers.

The IL-1β binding antibodies and fragments of the present invention alsoencompass heavy chain antibodies (HCAb). Exceptions to the H₂L₂structure of conventional antibodies occur in some isotypes of theimmunoglobulins found in camelids (camels, dromedaries and llamas;Hamers-Casterman et al., 1993 Nature 363: 446; Nguyen et al., 1998 J.Mol. Biol. 275: 413), wobbegong sharks (Nuttall et al., Mol. Immunol.38:313-26, 2001), nurse sharks (Greenberg et al., Nature 374:168-73,1995; Roux et al., 1998 Proc. Nat. Acad. Sci. USA 95: 11804), and in thespotted ratfish (Nguyen, et al., “Heavy-chain antibodies in Camelidae; acase of evolutionary innovation,” 2002 Immunogenetics 54(1): 39-47).These antibodies can apparently form antigen-binding regions using onlyheavy chain variable regions, in that these functional antibodies aredimers of heavy chains only (referred to as “heavy-chain antibodies” or“HCAbs”). Accordingly, some embodiments of the present IL-1β bindingantibodies and fragments may be heavy chain antibodies that specificallybind to IL-1β. For example, heavy chain antibodies that are a class ofIgG and devoid of light chains are produced by animals of the genusCamelidae which includes camels, dromedaries and llamas(Hamers-Casterman et al., Nature 363:446-448 (1993)). HCAbs have amolecular weight of about 95 kDa instead of the about 160 kDa molecularweight of conventional IgG antibodies. Their binding domains consistonly of the heavy-chain variable domains, often referred to as V_(HH) todistinguish them from conventional V_(H). Muyldermans et al., J. Mol.Recognit. 12:131-140 (1999). The variable domain of the heavy-chainantibodies is sometimes referred to as a nanobody (Cortez-Retamozo etal., Cancer Research 64:2853-57, 2004). A nanobody library may begenerated from an immunized dromedary as described in Conrath et al.,(Antimicrob Agents Chemother 45: 2807-12, 2001) or using recombinantmethods.

Since the first constant domain (C_(H1)) is absent (spliced out duringmRNA processing due to loss of a splice consensus signal), the variabledomain (V_(HH)) is immediately followed by the hinge region, the C_(H2)and the C_(H3) domains (Nguyen et al., Mol. Immunol. 36:515-524 (1999);Woolven et al., Immunogenetics 50:98-101 (1999)). Camelid V_(HH)reportedly recombines with IgG2 and IgG3 constant regions that containhinge, CH2, and CH3 domains and lack a CH1 domain (Hamers-Casterman etal., supra). For example, llama IgG1 is a conventional (H₂L₂) antibodyisotype in which V_(H) recombines with a constant region that containshinge, CH1, CH2 and CH3 domains, whereas the llama IgG2 and IgG3 areheavy chain-only isotypes that lack CH1 domains and that contain nolight chains.

Although the HCAbs are devoid of light chains, they have anantigen-binding repertoire. The genetic generation mechanism of HCAbs isreviewed in Nguyen et al. Adv. Immuntl 79:261-296 (2001) and Nguyen etal., Immunogenetics 54:39-47 (2002). Sharks, including the nurse shark,display similar antigen receptor-containing single monomeric V-domains.Irving et al., J. Immunol. Methods 248:31-45 (2001); Roux et al., Proc.Natl. Acad. Sci. USA 95:11804 (1998).

V_(HH)s comprise small intact antigen-binding fragments (for example,fragments that are about 15 kDa, 118-136 residues). Camelid V_(HH)domains have been found to bind to antigen with high affinity (Desmyteret al., J. Biol. Chem. 276:26285-90, 2001), with V_(HH) affinitiestypically in the nanomolar range and comparable with those of Fab andscFv fragments. V_(HH)s are highly soluble and more stable than thecorresponding derivatives of scFv and Fab fragments. V_(H) fragmentshave been relatively difficult to produce in soluble form, butimprovements in solubility and specific binding can be obtained whenframework residues are altered to be more V_(HH)-like. (See, forexample, Reichman et al., J Immunol Methods 1999, 231:25-38.) V_(HH)scarry amino acid substitutions that make them more hydrophilic andprevent prolonged interaction with BiP (immunoglobulin heavy-chainbinding protein), which normally binds to the H-chain in the EndoplasmicReticulum (ER) during folding and assembly, until it is displaced by theL-chain. Because of the V_(HH)s' increased hydrophilicity, secretionfrom the ER is improved.

Functional V_(HH)s may be obtained by proteolytic cleavage of HCAb of animmunized camelid, by direct cloning of V_(HH) genes from B-cells of animmunized camelid resulting in recombinant V_(HH)s, or from naive orsynthetic libraries. V_(HH)s with desired antigen specificity may alsobe obtained through phage display methodology. Using V_(HH)s in phagedisplay is much simpler and more efficient compared to Fabs or scFvs,since only one domain needs to be cloned and expressed to obtain afunctional antigen-binding fragment. Muyldermans, Biotechnol. 74:277-302(2001); Ghahroudi et al., FEBS Lett. 414:521-526 (1997); and van derLinden et al., J. Biotechnol. 80:261-270 (2000). Methods for generatingantibodies having camelid heavy chains are also described in U.S. PatentPublication Nos. 20050136049 and 20050037421.

Ribosome display methods may be used to identify and isolate scFv and/orV_(HH) molecules having the desired binding activity and affinity.Irving et al., J. Immunol. Methods 248:31-45 (2001). Ribosome displayand selection has the potential to generate and display large libraries(10¹⁴).

Other embodiments provide V_(HH)-like molecules generated through theprocess of camelisation, by modifying non-Camelidae V_(H)s, such ashuman V_(HH)s, to improve their solubility and prevent non-specificbinding. This is achieved by replacing residues on the V_(L)s side ofV_(H)s with V_(HH)-like residues, thereby mimicking the more solubleV_(HH) fragments. Camelised V_(H) fragments, particularly those based onthe human framework, are expected to exhibit a greatly reduced immuneresponse when administered in vivo to a patient and, accordingly, areexpected to have significant advantages for therapeutic applications.Davies et al., FEBS Lett. 339:285-290 (1994); Davies et al., ProteinEng. 9:531-537 (1996); Tanha et al., J. Biol. Chem. 276:24774-24780(2001); and Riechmann et al., Immunol. Methods 231:25-38 (1999).

A wide variety of expression systems are available for the production ofIL-1β fragments including Fab fragments, scFv, and V_(HH)s. For example,expression systems of both prokaryotic and eukaryotic origin may be usedfor the large-scale production of antibody fragments and antibody fusionproteins. Particularly advantageous are expression systems that permitthe secretion of large amounts of antibody fragments into the culturemedium.

Production of bispecific Fab-scFv (“bibody”) and trispecificFab-(scFv)(2) (“tribody”) are described in Schoonjans et al. (J Immunol.165:7050-57, 2000) and Willems et al. (J Chromatogr B Analyt TechnolBiomed Life Sci. 786:161-76, 2003). For bibodies or tribodies, a scFvmolecule is fused to one or both of the VL-CL (L) and VH-CH₁ (Fd)chains, e.g., to produce a tribody two scFvs are fused to C-term of Fabwhile in a bibody one scFv is fused to C-term of Fab. A “minibody”consisting of scFv fused to CH3 via a peptide linker (hingeless) or viaan IgG hinge has been described in Olafsen, et al., Protein Eng Des Sel.2004 April; 17(4):315-23.

Intrabodies are single chain antibodies which demonstrate intracellularexpression and can manipulate intracellular protein function (Biocca, etal., EMBO J. 9:101-108, 1990; Colby et al., Proc Natl Acad Sci USA.101:17616-21, 2004). Intrabodies, which comprise cell signal sequenceswhich retain the antibody construct in intracellular regions, may beproduced as described in Mhashilkar et al (EMBO J 14:1542-51, 1995) andWheeler et al. (FASEB J. 17:1733-5, 2003). Transbodies arecell-permeable antibodies in which a protein transduction domains (PTD)is fused with single chain variable fragment (scFv) antibodies Heng etal., (Med Hypotheses. 64:1105-8, 2005).

The IL-1β binding antibodies and fragments of the present invention alsoencompass antibodies that are SMIPs or binding domain immunoglobulinfusion proteins specific for target protein. These constructs aresingle-chain polypeptides comprising antigen binding domains fused toimmunoglobulin domains necessary to carry out antibody effectorfunctions. See e.g., WO03/041600, U.S. Patent publication 20030133939and US Patent Publication 20030118592.

The IL-1β binding antibodies and fragments of the present invention alsoencompass immunoadhesins. One or more CDRs may be incorporated into amolecule either covalently or noncovalently to make it an immunoadhesin.An immunoadhesin may incorporate the CDR(s) as part of a largerpolypeptide chain, may covalently link the CDR(s) to another polypeptidechain, or may incorporate the CDR(s) noncovalently. The CDRs disclosedherein permit the immunoadhesin to specifically bind to IL-1β.

The IL-1β binding antibodies and fragments of the present invention alsoencompass antibody mimics comprising one or more IL-1β binding portionsbuilt on an organic or molecular scaffold (such as a protein orcarbohydrate scaffold). Proteins having relatively definedthree-dimensional structures, commonly referred to as protein scaffolds,may be used as reagents for the design of antibody mimics. Thesescaffolds typically contain one or more regions which are amenable tospecific or random sequence variation, and such sequence randomizationis often carried out to produce libraries of proteins from which desiredproducts may be selected. For example, an antibody mimic can comprise achimeric non-immunoglobulin binding polypeptide having animmunoglobulin-like domain containing scaffold having two or moresolvent exposed loops containing a different CDR from a parent antibodyinserted into each of the loops and exhibiting selective bindingactivity toward a ligand bound by the parent antibody.Non-immunoglobulin protein scaffolds have been proposed for obtainingproteins with novel binding properties. (Tramontano et al., J. Mol.Recognit. 7:9, 1994; McConnell and Hoess, J. Mol. Biol. 250:460, 1995).Other proteins have been tested as frameworks and have been used todisplay randomized residues on alpha helical surfaces (Nord et al., Nat.Biotechnol. 15:772, 1997; Nord et al., Protein Eng. 8:601, 1995), loopsbetween alpha helices in alpha helix bundles (Ku and Schultz, Proc.Natl. Acad. Sci. USA 92:6552, 1995), and loops constrained by disulfidebridges, such as those of the small protease inhibitors (Markland etal., Biochemistry 35:8045, 1996; Markland et al., Biochemistry 35:8058,1996; Rottgen and Collins, Gene 164:243, 1995; Wang et al., J. Biol.Chem. 270:12250, 1995). Methods for employing scaffolds for antibodymimics are disclosed in U.S. Pat. No. 5,770,380 and US PatentPublications 2004/0171116, 2004/0266993, and 2005/0038229.

Preferred IL-1β antibodies or antibody fragments for use in accordancewith the invention generally bind to human IL-1β with high affinity(e.g., as determined with BIACORE), such as for example with anequilibrium binding dissociation constant (K_(D)) for IL-1β of about 10nM or less, about 5 nM or less, about 1 nM or less, about 500 pM orless, or more preferably about 250 pM or less, about 100 pM or less,about 50 pM or less, about 25 pM or less, about 10 pM or less, about 5pM or less, about 3 pM or less about 1 pM or less, about 0.75 pM orless, about 0.5 pM or less, or about 0.3 pM or less.

Antibodies or fragments of the present invention may, for example, bindto IL-1β with an IC₅₀ of about 10 nM or less, about 5 nM or less, about2 nM or less, about 1 nM or less, about 0.75 nM or less, about 0.5 nM orless, about 0.4 nM or less, about 0.3 nM or less, or even about 0.2 nMor less, as determined by enzyme linked immunosorbent assay (ELISA).Preferably, the antibody or antibody fragment of the present inventiondoes not cross-react with any target other than IL-1. For example, thepresent antibodies and fragments may bind to IL-1β, but do notdetectably bind to IL-1α, or have at least about 100 times (e.g., atleast about 150 times, at least about 200 times, or even at least about250 times) greater selectivity in its binding of IL-1β relative to itsbinding of IL-1α. Antibodies or fragments used according to theinvention may, in certain embodiments, inhibit IL-1β induced expressionof serum IL-6 in an animal by at least 50% (e.g., at least 60%, at least70%, or even at least 80%) as compared to the level of serum IL-6 in anIL-1β stimulated animal that has not been administered an antibody orfragment of the invention. Antibodies may bind IL-1β but permit orsubstantially permit the binding of the bound IL-1β ligand to IL-1receptor type I (IL-1RI). In contrast to many known IL-1β bindingantibodies that block or substantially interfere with binding of IL-1βto IL-1RI, the antibodies designated AB5 and AB7 (U.S. application Ser.No. 11/472,813) selectively bind to the IL-1β ligand, but permit thebinding of the bound IL-1β ligand to IL-1RI. For example, the antibodydesignated AB7 binds to an IL-1β epitope but still permits the boundIL-1β to bind to IL-1RI. In certain embodiments, the antibody maydecrease the affinity of interaction of bound IL-1β to bind to IL-1RI.Accordingly, the invention provides, in a related aspect, use of anIL-1β binding antibody or IL-1β binding antibody fragment that has atleast one of the aforementioned characteristics. Any of the foregoingantibodies, antibody fragments, or polypeptides of the invention can behumanized or human engineered, as described herein.

A variety of IL-1 (e.g., IL-1β) antibodies and fragments known in theart may be used according the methods provided herein, including forexample antibodies described in or derived using methods described inthe following patents and patent applications: U.S. Pat. No. 4,935,343;US 2003/0026806; US 2003/0124617; WO 2006/081139; WO 03/034984; WO95/01997; WO 02/16436; WO 03/010282; WO 03/073982, WO 2004/072116, WO2004/067568, EP 0 267 611 B1, EP 0 364 778 B1, and U.S. application Ser.No. 11/472,813. As a non-limiting example, antibodies AB5 and AB7 (U.S.application Ser. No. 11/472,813, WO2007/002261) may be used inaccordance with the invention. Variable region sequences of AB5 and AB7are as follows:

AB5

Light Chain

(SEQ ID NO: 3) DIQMTQTTSSLSASLGDRVTISCRASQDISNYLSWYQQKPDGTVKLLIYYTSKLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCLQGKM LPWTFGGGTKLEIKThe underlined sequences depict (from left to right) CDR1, 2 and 3.Heavy Chain

(SEQ ID NO: 4) QVTLKESGPGILKPSQTLSLTCSFSGFSLSTSGMGVGWIRQPSGKGLEWLAHIWWDGDESYNPSLKTQLTISKDTSRNQVFLKITSVDTVDTATYFCARNRYDPPWFVDWGQGTLVTVSSThe underlined sequences depict (from left to right) CDR1, 2 and 3.AB7Light Chain

(SEQ ID NO: 5) DIQMTQSTSSLSASVGDRVTITCRASQDISNYLSWYQQKPGKAVKLLI YYTS KLHSGVPSRFSGSGSGTDYTLTISSLQQEDFATYFCLQGKMLPW TFGQGTKLEIK The underlined sequences depict (from left to right) CDR1, 2 and 3.Heavy Chain

(SEQ ID NO: 6) QVQLQESGPGLVKPSQTLSLTCSFSGFSLSTSGMGVGWIRQPSGKGLEWLAHIWWDGDESYNPSLKSRLTISKDTSKNQVSLKITSVTAADTAVYFCARNRYDPPWFVDWGQGTLVTVSSThe underlined sequences depict (from left to right) CDR1, 2 and 3.

The antibodies and antibody fragments described herein can be preparedby any suitable method. Suitable methods for preparing such antibodiesand antibody fragments are known in the art. Other methods for preparingthe antibodies and antibody fragments are as described herein as part ofthe invention. The antibody, antibody fragment, or polypeptide of theinvention, as described herein, can be isolated or purified to anydegree. As used herein, an isolated compound is a compound that has beenremoved from its natural environment. A purified compound is a compoundthat has been increased in purity, such that the compound exists in aform that is more pure than it exists (i) in its natural environment or(ii) when initially synthesized and/or amplified under laboratoryconditions, wherein “purity” is a relative term and does not necessarilymean “absolute purity.”

Pharmaceutical Compositions

IL-1 (e.g., IL-1β) binding antibodies and antibody fragments for useaccording to the present invention can be formulated in compositions,especially pharmaceutical compositions, for use in the methods herein.Such compositions comprise a therapeutically or prophylacticallyeffective amount of an IL-1β binding antibody or antibody fragment ofthe invention in admixture with a suitable carrier, e.g., apharmaceutically acceptable agent. Typically, IL-1β binding antibodiesand antibody fragments of the invention are sufficiently purified foradministration to an animal before formulation in a pharmaceuticalcomposition.

Pharmaceutically acceptable agents include carriers, excipients,diluents, antioxidants, preservatives, coloring, flavoring and dilutingagents, emulsifying agents, suspending agents, solvents, fillers,bulking agents, buffers, delivery vehicles, tonicity agents, cosolvents,wetting agents, complexing agents, buffering agents, antimicrobials, andsurfactants.

Neutral buffered saline or saline mixed with albumin are exemplaryappropriate carriers. The pharmaceutical compositions can includeantioxidants such as ascorbic acid; low molecular weight polypeptides;proteins, such as serum albumin, gelatin, or immunoglobulins;hydrophilic polymers such as polyvinylpyrrolidone; amino acids such asglycine, glutamine, asparagine, arginine or lysine; monosaccharides,disaccharides, and other carbohydrates including glucose, mannose, ordextrins; chelating agents such as EDTA; sugar alcohols such as mannitolor sorbitol; salt-forming counterions such as sodium; and/or nonionicsurfactants such as Tween, pluronics, or polyethylene glycol (PEG). Alsoby way of example, suitable tonicity enhancing agents include alkalimetal halides (preferably sodium or potassium chloride), mannitol,sorbitol, and the like. Suitable preservatives include benzalkoniumchloride, thimerosal, phenethyl alcohol, methylparaben, propylparaben,chlorhexidine, sorbic acid and the like. Hydrogen peroxide also can beused as preservative. Suitable cosolvents include glycerin, propyleneglycol, and PEG. Suitable complexing agents include caffeine,polyvinylpyrrolidone, beta-cyclodextrin orhydroxy-propyl-beta-cyclodextrin. Suitable surfactants or wetting agentsinclude sorbitan esters, polysorbates such as polysorbate 80,tromethamine, lecithin, cholesterol, tyloxapal, and the like. Thebuffers can be conventional buffers such as acetate, borate, citrate,phosphate, bicarbonate, or Tris-HCl. Acetate buffer may be about pH4-5.5, and Tris buffer can be about pH 7-8.5. Additional pharmaceuticalagents are set forth in Remington's Pharmaceutical Sciences, 18thEdition, A. R. Gennaro, ed., Mack Publishing Company, 1990.

The composition can be in liquid form or in a lyophilized orfreeze-dried form and may include one or more lyoprotectants,excipients, surfactants, high molecular weight structural additivesand/or bulking agents (see for example U.S. Pat. Nos. 6,685,940,6,566,329, and 6,372,716). In one embodiment, a lyoprotectant isincluded, which is a non-reducing sugar such as sucrose, lactose ortrehalose. The amount of lyoprotectant generally included is such that,upon reconstitution, the resulting formulation will be isotonic,although hypertonic or slightly hypotonic formulations also may besuitable. In addition, the amount of lyoprotectant should be sufficientto prevent an unacceptable amount of degradation and/or aggregation ofthe protein upon lyophilization. Exemplary lyoprotectant concentrationsfor sugars (e.g., sucrose, lactose, trehalose) in the pre-lyophilizedformulation are from about 10 mM to about 400 mM. In another embodiment,a surfactant is included, such as for example, nonionic surfactants andionic surfactants such as polysorbates (e.g. polysorbate 20, polysorbate80); poloxamers (e.g. poloxamer 188); poly (ethylene glycol) phenylethers (e.g. Triton); sodium dodecyl sulfate (SDS); sodium laurelsulfate; sodium octyl glycoside; lauryl-, myristyl-, linoleyl-, orstearyl-sulfobetaine; lauryl-, linoleyl- or stearyl-sarcosine;linoleyl-, myristyl-, or cetyl-betaine; lauroamidopropyl-,cocamidopropyl-, linoleamidopropyl-, myristamidopropyl-, paImidopropyl-,or isostearamidopropyl-betaine (e.g. lauroamidopropyl);myristamidopropyl-, paImidopropyl-, orisostearamidopropyl-dimethylamine; sodium methyl cocoyl-, or disodiummethyl ofeyl-taurate; and the MONAQUAT™ series (Mona Industries, Inc.,Paterson, N.J.), polyethyl glycol, polypropyl glycol, and copolymers ofethylene and propylene glycol (e.g. Pluronics, PF68 etc). Exemplaryamounts of surfactant that may be present in the pre-lyophilizedformulation are from about 0.001-0.5%. High molecular weight structuraladditives (e.g. fillers, binders) may include for example, acacia,albumin, alginic acid, calcium phosphate (dibasic), cellulose,carboxymethylcellulose, carboxymethylcellulose sodium,hydroxyethylcellulose, hydroxypropylcellulose,hydroxypropylmethylcellulose, microcrystalline cellulose, dextran,dextrin, dextrates, sucrose, tylose, pregelatinized starch, calciumsulfate, amylose, glycine, bentonite, maltose, sorbitol, ethylcellulose,disodium hydrogen phosphate, disodium phosphate, disodium pyrosulfite,polyvinyl alcohol, gelatin, glucose, guar gum, liquid glucose,compressible sugar, magnesium aluminum silicate, maltodextrin,polyethylene oxide, polymethacrylates, povidone, sodium alginate,tragacanth microcrystalline cellulose, starch, and zein. Exemplaryconcentrations of high molecular weight structural additives are from0.1% to 10% by weight. In other embodiments, a bulking agent (e.g.,mannitol, glycine) may be included.

Compositions can be suitable for parenteral administration. Exemplarycompositions are suitable for injection or infusion into an animal byany route available to the skilled worker, such as intraarticular,subcutaneous, intravenous, intramuscular, intraperitoneal, intracerebral(intraparenchymal), intracerebroventricular, intramuscular, intraocular,intraarterial, intralesional, intrarectal, transdermal, oral, andinhaled routes. A parenteral formulation typically will be a sterile,pyrogen-free, isotonic aqueous solution, optionally containingpharmaceutically acceptable preservatives.

Examples of non-aqueous solvents are propylene glycol, polyethyleneglycol, vegetable oils such as olive oil, and injectable organic esterssuch as ethyl oleate. Aqueous carriers include water, alcoholic/aqueoussolutions, emulsions or suspensions, including saline and bufferedmedia. Parenteral vehicles include sodium chloride solution, Ringers'dextrose, dextrose and sodium chloride, lactated Ringer's, or fixedoils. Intravenous vehicles include fluid and nutrient replenishers,electrolyte replenishers, such as those based on Ringer's dextrose, andthe like. Preservatives and other additives may also be present, suchas, for example, anti-microbials, anti-oxidants, chelating agents, inertgases and the like. See generally, Remington's Pharmaceutical Science,16th Ed., Mack Eds., 1980, which is incorporated herein by reference.

Pharmaceutical compositions described herein can be formulated forcontrolled or sustained delivery in a manner that provides localconcentration of the product (e.g., bolus, depot effect) sustainedrelease and/or increased stability or half-life in a particular localenvironment. The invention contemplates that in certain embodiments suchcompositions may include a significantly larger amount of antibody orfragment in the initial deposit, while the effective amount of antibodyor fragment actually released and available at any point in time for isin accordance with the disclosure herein an amount much lower than theinitial deposit. The compositions can include the formulation of IL-1βbinding antibodies, antibody fragments, nucleic acids, or vectors of theinvention with particulate preparations of polymeric compounds such aspolylactic acid, polyglycolic acid, etc., as well as agents such as abiodegradable matrix, injectable microspheres, microcapsular particles,microcapsules, bioerodible particles beads, liposomes, and implantabledelivery devices that provide for the controlled or sustained release ofthe active agent which then can be delivered as a depot injection.Techniques for formulating such sustained- or controlled-delivery meansare known and a variety of polymers have been developed and used for thecontrolled release and delivery of drugs. Such polymers are typicallybiodegradable and biocompatible. Polymer hydrogels, including thoseformed by complexation of enantiomeric polymer or polypeptide segments,and hydrogels with temperature or pH sensitive properties, may bedesirable for providing drug depot effect because of the mild andaqueous conditions involved in trapping bioactive protein agents (e.g.,antibodies). See, for example, the description of controlled releaseporous polymeric microparticles for the delivery of pharmaceuticalcompositions in PCT Application Publication WO 93/15722.

Suitable materials for this purpose include polylactides (see, e.g.,U.S. Pat. No. 3,773,919), polymers of poly-(a-hydroxycarboxylic acids),such as poly-D-(−)-3-hydroxybutyric acid (EP 133,988A), copolymers ofL-glutamic acid and gamma ethyl-L-glutamate (Sidman et al., Biopolymers,22: 547-556 (1983)), poly (2-hydroxyethyl-methacrylate) (Langer et al.,J. Biomed. Mater. Res., 15: 167-277 (1981), and Langer, Chem. Tech., 12:98-105 (1982)), ethylene vinyl acetate, or poly-D(−)-3-hydroxybutyricacid. Other biodegradable polymers include poly(lactones),poly(acetals), poly(orthoesters), and poly(orthocarbonates).Sustained-release compositions also may include liposomes, which can beprepared by any of several methods known in the art (see, e.g., Eppsteinet al., Proc. Natl. Acad. Sci. USA, 82: 3688-92 (1985)). The carrieritself, or its degradation products, should be nontoxic in the targettissue and should not further aggravate the condition. This can bedetermined by routine screening in animal models of the target disorderor, if such models are unavailable, in normal animals.

Micro encapsulation of recombinant proteins for sustained release hasbeen performed successfully with human growth hormone (rhGH),interferon-(rhIFN-), interleukin-2, and MN rgp120. Johnson et al., Nat.Med., 2:795-799 (1996); Yasuda, Biomed. Ther., 27:1221-1223 (1993); Horaet al., Bio/Technology. 8:755-758 (1990); Cleland, “Design andProduction of Single Immunization Vaccines Using PolylactidePolyglycolide Microsphere Systems,” in Vaccine Design The Subunit andAdjuvant Approach, Powell and Newman, eds, (Plenum Press: New York,1995), pp. 439-462; WO 97/03692, WO 96/40072, WO 96/07399; and U.S. Pat.No. 5,654,010. The sustained-release formulations of these proteins weredeveloped using polylactic-coglycolic acid (PLGA) polymer due to itsbiocompatibility and wide range of biodegradable properties. Thedegradation products of PLGA, lactic and glycolic acids can be clearedquickly within the human body. Moreover, the degradability of thispolymer can be depending on its molecular weight and composition. Lewis,“Controlled release of bioactive agents from lactide/glycolide polymer,”in: M. Chasin and R. Langer (Eds.), Biodegradable Polymers as DrugDelivery Systems (Marcel Dekker: New York, 1990), pp. 1-41. Additionalexamples of sustained release compositions include, for example, EP58,481A, U.S. Pat. No. 3,887,699, EP 158,277A, Canadian Patent No.1176565, U. Sidman et al., Biopolymers 22, 547 [1983], R. Langer et al.,Chem. Tech. 12, 98 [1982], Sinha et al., J. Control. Release 90, 261[2003], Zhu et al., Nat. Biotechnol. 18, 24 [2000], and Dai et al.,Colloids Surf B Biointerfaces 41, 117 [2005].

Bioadhesive polymers are also contemplated for use in or withcompositions of the present invention. Bioadhesives are synthetic andnaturally occurring materials able to adhere to biological substratesfor extended time periods. For example, Carbopol and polycarbophil areboth synthetic cross-linked derivatives of poly(acrylic acid).Bioadhesive delivery systems based on naturally occurring substancesinclude for example hyaluronic acid, also known as hyaluronan.Hyaluronic acid is a naturally occurring mucopolysaccharide consistingof residues of D-glucuronic and N-acetyl-D-glucosamine. Hyaluronic acidis found in the extracellular tissue matrix of vertebrates, including inconnective tissues, as well as in synovial fluid and in the vitreous andaqueous humour of the eye. Esterified derivatives of hyaluronic acidhave been used to produce microspheres for use in delivery that arebiocompatible and biodegrable (see for example, Cortivo et al.,Biomaterials (1991) 12:727-730; European Publication No. 517,565;International Publication No. WO 96/29998; Mum et al., J. ControlledRel. (1994) 29:133-141). Exemplary hyaluronic acid containingcompositions of the present invention comprise a hyaluronic acid esterpolymer in an amount of approximately 0.1% to about 40% (w/w) of anIL-1β binding antibody or fragment to hyaluronic acid polymer.

Both biodegradable and non-biodegradable polymeric matrices can be usedto deliver compositions in accordance with the invention, and suchpolymeric matrices may comprise natural or synthetic polymers.Biodegradable matrices are preferred. The period of time over whichrelease occurs is based on selection of the polymer. Typically, releaseover a period ranging from between a few hours and three to twelvemonths is most desirable. Exemplary synthetic polymers which can be usedto form the biodegradable delivery system include: polymers of lacticacid and glycolic acid, polyamides, polycarbonates, polyalkylenes,polyalkylene glycols, polyalkylene oxides, polyalkylene terepthalates,polyvinyl alcohols, polyvinyl ethers, polyvinyl esters, poly-vinylhalides, polyvinylpyrrolidone, polyglycolides, polysiloxanes,polyanhydrides, polyurethanes and co-polymers thereof, poly(butic acid),poly(valeric acid), alkyl cellulose, hydroxyalkyl celluloses, celluloseethers, cellulose esters, nitro celluloses, polymers of acrylic andmethacrylic esters, methyl cellulose, ethyl cellulose, hydroxypropylcellulose, hydroxy-propyl methyl cellulose, hydroxybutyl methylcellulose, cellulose acetate, cellulose propionate, cellulose acetatebutyrate, cellulose acetate phthalate, carboxylethyl cellulose,cellulose triacetate, cellulose sulphate sodium salt, poly(methylmethacrylate), poly(ethyl methacrylate), poly(butylmethacrylate),poly(isobutyl methacrylate), poly(hexylmethacrylate), poly(isodecylmethacrylate), poly(lauryl methacrylate), poly(phenyl methacrylate),poly(methyl acrylate), poly(isopropyl acrylate), poly(isobutylacrylate), poly(octadecyl acrylate), polyethylene, polypropylene,poly(ethylene glycol), poly(ethylene oxide), poly(ethyleneterephthalate), poly(vinyl alcohols), polyvinyl acetate, poly vinylchloride, polystyrene and polyvinylpyrrolidone. Exemplary naturalpolymers include alginate and other polysaccharides including dextranand cellulose, collagen, chemical derivatives thereof (substitutions,additions of chemical groups, for example, alkyl, alkylene,hydroxylations, oxidations, and other modifications routinely made bythose skilled in the art), albumin and other hydrophilic proteins, zeinand other prolamines and hydrophobic proteins, copolymers and mixturesthereof. In general, these materials degrade either by enzymatichydrolysis or exposure to water in vivo, by surface or bulk erosion. Thepolymer optionally is in the form of a hydrogel (see for example WO04/009664, WO 05/087201, Sawhney, et al., Macromolecules, 1993, 26,581-587) that can absorb up to about 90% of its weight in water andfurther, optionally is cross-linked with multi-valent ions or otherpolymers.

Delivery systems also include non-polymer systems that are lipidsincluding sterols such as cholesterol, cholesterol esters and fattyacids or neutral fats such as mono- di- and tri-glycerides; hydrogelrelease systems; silastic systems; peptide based systems; wax coatings;compressed tablets using conventional binders and excipients; partiallyfused implants; and the like. Specific examples include, but are notlimited to: (a) erosional systems in which the product is contained in aform within a matrix such as those described in U.S. Pat. Nos.4,452,775, 4,675,189 and 5,736,152 and (b) diffusional systems in whicha product permeates at a controlled rate from a polymer such asdescribed in U.S. Pat. Nos. 3,854,480, 5,133,974 and 5,407,686.Liposomes containing the product may be prepared by methods knownmethods, such as for example (DE 3,218,121; Epstein et al., Proc. Natl.Acad. Sci. USA, 82: 3688-3692 (1985); Hwang et al., Proc. Natl. Acad.Sci. USA, 77: 4030-4034 (1980); EP 52,322; EP 36,676; EP 88,046; EP143,949; EP 142,641; Japanese patent application 83-118008; U.S. Pat.Nos. 4,485,045 and 4,544,545; and EP 102,324).

A pharmaceutical composition comprising an IL-1β binding antibody orfragment can be formulated for inhalation, such as for example, as a drypowder. Inhalation solutions also can be formulated in a liquefiedpropellant for aerosol delivery. In yet another formulation, solutionsmay be nebulized. Additional pharmaceutical composition for pulmonaryadministration include, those described, for example, in PCT ApplicationPublication WO 94/20069, which discloses pulmonary delivery ofchemically modified proteins. For pulmonary delivery, the particle sizeshould be suitable for delivery to the distal lung. For example, theparticle size can be from 1 μm to 5 μm; however, larger particles may beused, for example, if each particle is fairly porous.

Certain formulations containing IL-1β binding antibodies or antibodyfragments can be administered orally. Formulations administered in thisfashion can be formulated with or without those carriers customarilyused in the compounding of solid dosage forms such as tablets andcapsules. For example, a capsule can be designed to release the activeportion of the formulation at the point in the gastrointestinal tractwhen bioavailability is maximized and pre-systemic degradation isminimized. Additional agents can be included to facilitate absorption ofa selective binding agent. Diluents, flavorings, low melting pointwaxes, vegetable oils, lubricants, suspending agents, tabletdisintegrating agents, and binders also can be employed.

Another preparation can involve an effective quantity of an IL-1βbinding antibody or fragment in a mixture with non-toxic excipientswhich are suitable for the manufacture of tablets. By dissolving thetablets in sterile water, or another appropriate vehicle, solutions canbe prepared in unit dose form. Suitable excipients include, but are notlimited to, inert diluents, such as calcium carbonate, sodium carbonateor bicarbonate, lactose, or calcium phosphate; or binding agents, suchas starch, gelatin, or acacia; or lubricating agents such as magnesiumstearate, stearic acid, or talc.

Suitable and/or preferred pharmaceutical formulations can be determinedin view of the present disclosure and general knowledge of formulationtechnology, depending upon the intended route of administration,delivery format, and desired dosage. Regardless of the manner ofadministration, an effective dose can be calculated according to patientbody weight, body surface area, or organ size. Further refinement of thecalculations for determining the appropriate dosage for treatmentinvolving each of the formulations described herein are routinely madein the art and is within the ambit of tasks routinely performed in theart. Appropriate dosages can be ascertained through use of appropriatedose-response data.

Additional formulations will be evident in light of the presentdisclosure, including formulations involving IL-1β binding antibodiesand fragments in combination with one or more other therapeutic agents.For example, in some formulations, an IL-1β binding antibody, antibodyfragment, nucleic acid, or vector of the invention is formulated with asecond inhibitor of an IL-1 signaling pathway Representative secondinhibitors include, but are not limited to, antibodies, antibodyfragments, peptides, polypeptides, compounds, nucleic acids, vectors andpharmaceutical compositions, such as, for example, those described inU.S. Pat. No. 6,899,878, US 2003022869, US 20060094663, US 20050186615,US 20030166069, WO/04022718, WO/05084696, WO/05019259. For example, acomposition may comprise an IL-1β binding antibody, antibody fragment,nucleic acid, or vector of the invention in combination with an IL-1βbinding antibody, fragment, or a nucleic acid or vector encoding such anantibody or fragment.

The pharmaceutical compositions can comprise IL-1β binding antibodies orfragments in combination with other active agents. Such combinations arethose useful for their intended purpose. The combinations which are partof this invention can be IL-1β antibodies and fragments, such as forexample those described herein, and at least one additional agentselected from the lists below. The active agents set forth below areillustrative for purposes and not intended to be limited. Thecombination can also include more than one additional agent, e.g., twoor three additional agents if the combination is such that the formedcomposition can perform its intended function.

The invention further contemplates that pharmaceutical compositionscomprising one or more other active agents may be administeredseparately from the IL-1β binding antibodies or fragments, and suchseparate administrations may be performed at the same point or differentpoints in time, such as for example the same or different days.Administration of the other active agents may be according to standardmedical practices known in the art, or the administration may bemodified (e.g., longer intervals, smaller dosages, delayed initiation)when used in conjunction with administration of IL-1β binding antibodiesor fragments, such as disclosed herein.

Active agents or combinations with the present antibodies or fragmentsinclude a non-steroidal anti-inflammatory drug (NSAID) such as aspirin,ibuprofen, and other propionic acid derivatives (alminoprofen,benoxaprofen, bucloxic acid, carprofen, fenbufen, fenoprofen, fluprofen,flurbiprofen, indoprofen, ketoprofen, miroprofen, naproxen, oxaprozin,pirprofen, pranoprofen, suprofen, tiaprofenic acid, and tioxaprofen),acetic acid derivatives (indomethacin, acemetacin, alclofenac, clidanac,diclofenac, fenclofenac, fenclozic acid, fentiazac, fuirofenac,ibufenac, isoxepac, oxpinac, sulindac, tiopinac, tolmetin, zidometacin,and zomepirac), fenamic acid derivatives (flufenamic acid, meclofenamicacid, mefenamic acid, niflumic acid and tolfenamic acid),biphenylcarboxylic acid derivatives (diflunisal and flufenisal), oxicams(isoxicam, piroxicam, sudoxicam and tenoxican), salicylates (acetylsalicylic acid, sulfasalazine) and the pyrazolones (apazone,bezpiperylon, feprazone, mofebutazone, oxyphenbutazone, phenylbutazone).Other combinations include cyclooxygenase-2 (COX-2) inhibitors. Otheractive agents for combination include steroids such as prednisolone,prednisone, methylprednisolone, betamethasone, dexamethasone, orhydrocortisone. Such a combination may be especially advantageous, sinceone or more side-effects of the steroid can be reduced or eveneliminated by tapering the steroid dose required when treating patientsin combination with the present antibodies and fragments.

Alternatively or in addition, therapeutic treatment with at least one ormore additional active agents may be used which may act via differentmodes of action: 1) sulfonylureas (e.g., chlorpropamide, tolazamide,acetohexamide, tolbutamide, glyburide, glimepiride, glipizide) and/ormeglitinides (e.g., repaglinide, nateglinide) that essentially stimulateinsulin secretion; 2) biguanides (e.g., metformin) act by promotingglucose utilization, reducing hepatic glucose production and diminishingintestinal glucose output; 3) alpha-glucosidase inhibitors (e.g.,acarbose, miglitol) slow down carbohydrate digestion and consequentlyabsorption from the gut and reduce postprandial hyperglycemia; 4)thiazolidinediones (e.g., troglitazone, pioglitazone, rosiglitazone,glipizide, balaglitazone, rivoglitazone, netoglitazone, troglitazone,englitazone, AD 5075, T 174, YM 268, R 102380, NC 2100, NIP 223, NIP221, MK 0767, ciglitazone, adaglitazone, CLX 0921, darglitazone, CP92768, BM 152054) that enhance insulin action, thus promoting glucoseutilization in peripheral tissues; 5) glucagon-like-peptides includingDPP4 inhibitors (e.g., sitagliptin); and 6) insulin, which stimulatestissue glucose utilization and inhibits hepatic glucose output.Glucagon-like peptide-1 (GLP-1), DPP-IV-resistant analogues (incretinmimetics), DPP-IV inhibitors, insulin, insulin analogues, PPAR gammaagonists, dual-acting PPAR agonists, GLP-1 agonists or analogues, PTP1Binhibitors, SGLT inhibitors, insulin secretagogues, RXR agonists,glycogen synthase kinase-3 inhibitors, insulin sensitizers, immunemodulators, beta-3 adrenergic receptor agonists, Pan-PPAR agonists,11beta-HSD1 inhibitors, amylin analogues, biguanides, alpha-glucosidaseinhibitors, meglitinides, thiazolidinediones, sulfonylureas and the likealso may be used as the other active agent(s) (see for example Nathan,2006, N. Engl. J. Med. 355:2477-2480; Kahn et al., 2006, N. Engl. J.Med. 355:2427-2443). In yet another embodiment, the active agent may bean HMG Co-A reductase inhibitor (e.g., statins).

It is further contemplated that an anti-IL-1β antibody or fragmentadministered to a subject in accordance with the invention may beadministered in combination with treatment with at least one additionalactive agent, such as for example any of the aforementioned activeagents. In one embodiment, treatment with the at least one active agentis maintained. In another embodiment, treatment with the at least oneactive agent is reduced or discontinued (e.g., when the subject isstable), while treatment with the anti-IL-1β antibody or fragment ismaintained at a constant dosing regimen. In another embodiment,treatment with the at least one active agent is reduced or discontinued(e.g., when the subject is stable), and treatment with the anti-IL-1βantibody or fragment is reduced (e.g., lower dose, less frequent dosing,shorter treatment regimen). In another embodiment, treatment with the atleast one active agent is reduced or discontinued (e.g., when thesubject is stable), and treatment with the anti-IL-1β antibody orfragment is increased (e.g., higher dose, more frequent dosing, longertreatment regimen). In yet another embodiment, treatment with the atleast one active agent is maintained and treatment with the anti-IL-1βantibody or fragment is reduced or discontinued (e.g., lower dose, lessfrequent dosing, shorter treatment regimen). In yet another embodiment,treatment with the at least one active agent and treatment with theanti-IL-1β antibody or fragment are reduced or discontinued (e.g., lowerdose, less frequent dosing, shorter treatment regimen)

The pharmaceutical compositions used in the invention may include atherapeutically effective amount or a prophylactically effective amountof the IL-1β binding antibodies or fragments. A therapeuticallyeffective amount refers to an amount effective, at dosages and forperiods of time necessary, to achieve the desired therapeutic result. Atherapeutically effective amount of the antibody or antibody portion mayvary according to factors such as the disease state, age, sex, andweight of the individual, and the ability of the antibody or antibodyportion to elicit a desired response in the individual. Atherapeutically effective amount is also one in which any toxic ordetrimental effects of the antibody or antibody portion are outweighedby the therapeutically beneficial effects. A prophylactically effectiveamount refers to an amount effective, at dosages and for periods of timenecessary, to achieve the desired prophylactic result.

A therapeutically or prophylactically effective amount of apharmaceutical composition comprising an IL-1β binding antibody orfragment will depend, for example, upon the therapeutic objectives suchas the indication for which the composition is being used, the route ofadministration, and the condition of the subject. Pharmaceuticalcompositions are administered in a therapeutically or prophylacticallyeffective amount to treat an IL-1 related condition. A “therapeuticallyor prophylactically effective amount” of an IL-1β binding antibody orfragment, of the invention is that amount which can treat or prevent oneor more symptoms of an IL-1 related disease in a subject, as disclosedherein.

Methods of Use

Anti-IL-1β antibodies in an effective amount may be used in the presentinvention for the treatment and/or prevention of Type 1 diabetes, Type 2diabetes, obesity, hyperglycemia, hyperinsulinemia, insulin resistanceand disease states and conditions characterized by insulin resistance.Such methods may be used to treat a mammalian subject (e.g., human)suffering from Type 2 diabetes, Type 1 diabetes, obesity, hyperglycemia,hyperinsulinemia, insulin resistance and disease states and conditionscharacterized by insulin resistance or to prevent occurrence of the samein an at risk subject.

The terms “prevention”, “prevent”, “preventing”, “suppression”,“suppress”, “suppressing”, “inhibit” and “inhibition” as used hereinrefer to a course of action (such as administering a compound orpharmaceutical composition) initiated in a manner (e.g., prior to theonset of a clinical symptom of a disease state or condition) so as toprevent, suppress or reduce, either temporarily or permanently, theonset of a clinical manifestation of the disease state or condition.Such preventing, suppressing or reducing need not be absolute to beuseful.

The terms “treatment”, “treat” and “treating” as used herein refers acourse of action (such as administering a compound or pharmaceuticalcomposition) initiated after the onset of a clinical symptom of adisease state or condition so as to eliminate, reduce, suppress orameliorate, either temporarily or permanently, a clinical manifestationor progression of the disease state or condition. Such treating need notbe absolute to be useful.

The term “in need of treatment” as used herein refers to a judgment madeby a caregiver that a patient requires or will benefit from treatment.This judgment is made based on a variety of factors that are in therealm of a caregiver's expertise, but that includes the knowledge thatthe patient is ill, or will be ill, as the result of a condition that istreatable by a method or compound of the disclosure.

The term “in need of prevention” as used herein refers to a judgmentmade by a caregiver that a patient requires or will benefit fromprevention. This judgment is made based on a variety of factors that arein the realm of a caregiver's expertise, but that includes the knowledgethat the patient will be ill or may become ill, as the result of acondition that is preventable by a method or compound of the disclosure.

The term “therapeutically effective amount” as used herein refers to anamount of a compound (e.g., antibody), either alone or as a part of apharmaceutical composition, that is capable of having any detectable,positive effect on any symptom, aspect, or characteristics of a diseasestate or condition when administered to a patient (e.g., as one or moredoses). Such effect need not be absolute to be beneficial.

The term “insulin resistance” as used herein refers to a condition wherea normal amount of insulin is unable to produce a normal physiologicalor molecular response. In some cases, a hyper-physiological amount ofinsulin, either endogenously produced or exogenously added, is able toovercome the insulin resistance in whole or in part and produce abiologic response.

Anti-IL-1β antibodies or fragments may be administered to a human in aneffective amount for the treatment and/or prevention of Type 2 diabetes,Type 1 diabetes, obesity, hyperglycemia, hyperinsulinemia, insulinresistance and/or disease states and conditions characterized by insulinresistance. Other diseases or conditions contemplated for treatment withanti-IL-1β antibodies or fragments according to the present inventioninclude pre-diabetes, dyslipidemia, hyperlipidemia, hypertension,Metabolic Syndrome and Sickness Behavior. The invention furthercontemplates methods of using such antibodies or fragments to decreasethe incidence or severity, or stabilize, complications or conditionsassociated with Type 2 diabetes, such as for example, retinopathy, renalfailure, cardiovascular disease (e.g., atherosclerosis, peripheralvascular disease), and wound healing (e.g., diabetic ulcer).

In addition, the invention further contemplates the use of IL-1βantibodies and fragments as described herein to reduce the level ofC-reactive protein (CRP) in a subject. CRP is an acute phase proteinthat is produced predominantly by hepatocytes under the influence ofcytokines such as IL-1, IL-6, and tumor necrosis factor (TNF). Based onthe 2007 electronic version of the internal medicine textbook UpToDate®,despite a lack of specificity for the cause of inflammation (e.g.,infection, chronic renal disease, auto-inflammatory disease, cancer),data from more than 30 epidemiologic studies have shown a significantassociation between elevated serum or plasma concentrations of CRP andthe prevalence of underlying atherosclerosis, the risk of recurrentcardiovascular events among patients with established disease, and theincidence of first cardiovascular events among individuals at risk foratherosclerosis. In addition, the interplay of primary renal disease,the resultant kidney failure with its oxidative stress andpost-synthetic protein modifications, dialysis with the associatedcontaminants and effect of the dialysis membrane on serum proteins, andthe infections associated with repeated access site entry and subsequentsystemic infections leads these patients to an excessive load ofinflammatory stimuli. As the serum creatinine clearance levels fall withthe worsening renal function there is a proportional rise in of seruminflammatory mediators (e.g., cytokines TNF, IL-6, IL-1) as well asevidence of the body attempting to combat this situation with increased,but inefficient, production of IL-1 RA and IL-10, anti-inflammatorymediators. This inflammatory state in chronic renal failure patientsleads to atherosclerotic plaque instability due to direct triggering ofapoptosis of vascular smooth muscle cells. The consequence of cytokineelevation leads to one of the top two major mortalities in thesepatients—a remarkable increase in cardiovascular deaths from myocardialinfarctions and strokes. A direct illustration of this increased risk isseen with evaluation of patient's CRP levels; when divided intoquartiles of CRP values, the group with the highest CRP values has a12-month mortality rate of approximately 35%. Thus, the presentinvention discloses the use of an IL-1β antibody or fragment as providedherein to reduce CRP levels in such patients (e.g., subjects sufferingfrom renal disease). The reduction in CRP levels in a subject asdescribed herein is a suitable means to achieve a correspondingproportional decrease in cardiovascular morbidity and mortality.

In one embodiment, the anti-IL-1β antibody or fragment is administeredto a subject with at least one of the aforementioned diseases,conditions, or complications and the subject also receives at least oneother medically accepted treatment (e.g, medication, drug, therapeutic,active agent) for the disease, condition or complication. In anotherembodiment, the at least one other medically accepted treatment for thedisease, condition or complication is reduced or discontinued (e.g.,when the subject is stable), while treatment with the anti-IL-1βantibody or fragment is maintained at a constant dosing regimen. Inanother embodiment, the at least one other medically accepted treatmentfor the disease, condition or complication is reduced or discontinued(e.g., when the subject is stable), and treatment with the anti-IL-1βantibody or fragment is reduced (e.g., lower dose, less frequent dosing,shorter treatment regimen). In another embodiment, the at least oneother medically accepted treatment for the disease, condition orcomplication is reduced or discontinued (e.g., when the subject isstable), and treatment with the anti-IL-1β antibody or fragment isincreased (e.g., higher dose, more frequent dosing, longer treatmentregimen). In yet another embodiment, the at least one other medicallyaccepted treatment for the disease, condition or complication ismaintained and treatment with the anti-IL-1β antibody or fragment isreduced or discontinued (e.g., lower dose, less frequent dosing, shortertreatment regimen). In yet another embodiment, the at least one othermedically accepted treatment for the disease, condition or complicationand treatment with the anti-IL-1β antibody or fragment are reduced ordiscontinued (e.g., lower dose, less frequent dosing, shorter treatmentregimen)

In preferred methods of treating or preventing the aforementioneddiseases or conditions (e.g., Type 1 diabetes, Type 2 diabetes,hyperglycemia, hyperinsulinemia, obesity, insulin resistance) anti-IL-1βantibody or fragment thereof is administered to the human subjectaccording to the aforementioned numbers of doses, amounts per doseand/or intervals between dosing. Alternatively, the anti-IL-1β antibodyor fragment may be administered as one or more initial doses of theaforementioned amounts that are lower than one or more subsequent doseamounts. By providing the initial dose(s) in a lower amount, theeffectiveness and/or tolerability of the treatment may be enhanced. Forexample, in a non-limiting embodiment of the invention, one or moreinitial doses (e.g., 1, 2, 3, 4, 5) of an amount of antibody orfragment≦1 mg/kg (e.g., ≦0.9 mg/kg, ≦0.8 mg/kg, ≦0.7 mg/kg, ≦0.6 mg/kg,≦0.5 mg/kg, ≦0.4 mg/kg, ≦0.3 mg/kg, ≦0.2 mg/kg, ≦0.1 mg/kg, ≦0.05 mg/kg,≦0.03 mg/kg, ≦0.01 mg/kg) may be administered, followed by one or moresubsequent doses in an amount greater than the initial dose(s) (e.g.,≧0.01 mg/kg, ≧0.03 mg/kg, ≧0.1 mg/kg, ≧0.3 mg/kg≧0.5 mg/kg, ≧0.6 mg/kg,≧0.7 mg/kg, ≧0.8 mg/kg, ≧0.9 mg/kg, ≧1.0 mg/kg, ≧1.5 mg/kg, ≧2 mg/kg,≧2.5 mg/kg, ≧3 mg/kg, ≧3.5 mg/kg, ≧4 mg/kg, ≧4.5 mg/kg, ≧5 mg/kg). Theinvention contemplates that each dose of antibody or fragment may beadministered at one or more sites.

Methods of treating or preventing a disease or condition in accordancewith the present invention may use a pre-determined or “routine”schedule for administration of the antibody or fragment. As used hereina routine schedule refers to a predetermined designated period of timebetween dose administrations. The routine schedule may encompass periodsof time which are identical or which differ in length, as long as theschedule is predetermined. Any particular combination would be coveredby the routine schedule as long as it is determined ahead of time thatthe appropriate schedule involves administration on a certain day.

The invention further contemplates that IL-1β antibodies or fragmentsused in accordance with the methods provided herein, may be administeredin conjunction with more traditional treatment methods andpharmaceutical compositions (e.g., active agents). Such compositions,may include for example, DPP-IV inhibitors, insulin, insulin analogues,PPAR gamma agonists, dual-acting PPAR agonists, GLP-1 agonists oranalogues, PTP1B inhibitors, SGLT inhibitors, insulin secretagogues, RXRagonists, glycogen synthase kinase-3 inhibitors, insulin sensitizers,immune modulators, beta-3 adrenergic receptor agonists, Pan-PPARagonists, 11beta-HSD1 inhibitors, amylin analogues, biguanides,alpha-glucosidase inhibitors, meglitinides, thiazolidinediones,sulfonylureas and the like (see for example Nathan, 2006, N. Engl. J.Med. 355:2477-2480; Kahn et al., 2006, N. Engl. J. Med. 355:2427-2443).In certain embodiments, the antibodies and fragments used in accordancewith the invention may prevent or delay the need for additionaltreatment methods or pharmaceutical compositions. In other embodiments,the antibodies or fragments may reduce the amount, frequency or durationof additional treatment methods or pharmaceutical compositions.

Alternatively, methods of treating or preventing a disease or conditionin accordance with the present invention may use a schedule foradministration of the antibody or fragment that is based upon thepresence of disease symptoms and/or changes in any of the assessmentsherein (e.g., HbA1c, fasting blood sugar levels, OGTT, glucose/insulinC-peptide AUC, use of diabetes medication, insulin sensitivity, serumcytokine levels, CRP levels, quality of life measurements, BMIimprovement) as a means to determine when to administer one or moresubsequent doses. Similar, this approach may be used as a means todetermine whether a subsequent dose should be increased or decreased,based upon the effect of a previous dose.

Diagnosis of such diseases or conditions in patients, or alternativelythe risk for developing such diseases or conditions may be according tostandard medical practices known in art. Following administration of ananti-IL-1β antibodies or fragment, clinical assessments for a treatmentor preventative effect on the aforementioned diseases and conditions arewell known in the art and may be used as a means to monitor theeffectiveness of methods of the invention.

For example, response to treatment of Type 2 diabetes may be assessedbased on a primary efficacy endpoint of improvement in hemoglobin A1c(HbA1c, see for example Reynolds et al., BMJ, 333(7568):586-589, 2006).Improvements in HbA1c that are indicative of therapeutic efficacy mayvary depending on the initial baseline measurement in a patient, with alarger decrease often corresponding to a higher initial baseline and asmaller decrease often corresponding to a lower initial baseline. In oneaspect of the invention, the method should result in an HbA1c decreaseof at least about 0.5% (e.g., at least about 0.5%, at least about 1%, atleast about 1.5%, at least about 2%, at least about 2.5%, at least about3%, at least about 3.5%, at least about 4% or more) compared withpre-dose levels.

One or more of the following secondary endpoints also may be determinedin order to assess efficacy of the treatment, such as for examplefasting blood sugar (e.g., glucose) levels (e.g., decrease to ≦130,≦125, ≦120, ≦115, ≦110, ≦105, ≦100; alternatively decreaseof >20%, >30%, >40%, >50%, >60%, >70%, >80%, >90%, >95% compared topre-dose levels), 120 minute oral glucose tolerance test (OGTT) (e.g.,≦200, ≦190, ≦180, ≦170, ≦160, ≦150, ≦140), glucose/insulin C-peptide AUC(e.g., >25%, >50%, >60%, >70%, >80%, >90%, >100% increase frompre-treatment), reduction in diabetes medication (e.g., insulin, oralhypoglycemic agent), improvement in insulin sensitivity, serum cytokinelevels (e.g., normalization), CRP levels (e.g., decrease of ≧0.2, ≧0.4,≧0.6, ≧0.8, ≧1.0, ≧1.4, ≧1.8, ≧2.2, ≧2.6, ≧3.0 mg/L; alternatively adecrease of >20%, >30%, >40%, >50%, >60%, >70%, >80%, >90%, >95% frompre-treatment) quality of life measurements, BMI improvement (reductionof 1%, 3%, 5%), pharmacokinetics, and the like (Saudek, et al., JAMA,295:1688-97, 2006; Pfutzner et al., Diabetes Technol Ther. 8:28-36,2006; Norberg, et al., J Intern Med. 260:263-71, 2006).

Similarly, assessment of efficacy for other diseases or conditions mayuse one or more of the aforementioned endpoints and/or others known inthe art. For example, the effect on hyperglycemia can be assessed bymeasuring fasting blood sugar (i.e., glucose) levels, the effect onhyperinsulinemia may be assessed by measuring insulin levels and/orC-peptide levels, the effect on obesity may be assessed by measuringweight and/or BMI, and the effect on insulin resistance may be assessedby OGTT.

Alternatively, or in addition, subjects treated in accordance with theinvention may experience a decrease in the triglyceride level in theblood of the subject of at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%,45%, 50%, 55%, 60%, 65%, 70%, or more from the pre-treatment level.Alternatively, or in addition, subjects treated in accordance with theinvention may experience a decrease in the level of free fatty acids ofat least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, ormore from the pre-treatment level.

EXAMPLES

The following examples are intended merely to further illustrate thepractice of the present invention, but should not be construed as in anyway limiting its scope. The disclosures of all patent and scientificliteratures cited within are hereby expressly incorporated in theirentirety by reference.

Example 1 Inhibition of IL-1β Using a High Affinity IL-1β Antibody in anIn Vitro Cell Based Assay, with IL-1 Induced Production of IL-8 as aRead-Out

The inhibitory effect of the IL-1β-specific antibody was compared to anon-antibody inhibitor of the IL-1 pathway, Kineret® (anakinra), whichis a recombinant IL-1 receptor antagonist. Fresh, heparinized peripheralblood was collected from healthy donors. 180 μl of whole blood wasplated in a 96-well plate and incubated with various concentrations ofthe antibody AB7 (U.S. application Ser. No. 11/472,813, WO 2007/002261)and 100 pM rhJL-113. For Kineret®-treated samples, Kineret® and rhIL-1βwere combined 1:1 prior to mixing with blood. Samples were incubated for6 hours at 37° C. with 5% CO₂. Whole blood cells were then lysed with 50μl 2.5% Triton X-100. The concentration of interleukin-8 (IL-8) incleared lysates was assayed by ELISA (Quantikine human IL-8 ELISA kit,R&D Systems) according to manufacturer's instructions. IL-8concentrations in AB7 and Kineret® treated samples were compared to acontrol sample treated with anti-KLH control. The results are depictedin FIG. 1 and summarized in Table 6. IC₅₀ is the concentration ofantibody required to inhibit 50% of IL-8 released by IL-1β stimulation.

TABLE 1 IC₅₀ (pM) AB7  1.9 pM Kineret ® 53.4 pM

These results demonstrate the in vitro potency of AB7, as measured byinhibition of IL-1β stimulated release of IL-8. These results showinggreater potency compared with Kineret® indicate that the antibody willhave IL-1β inhibitory efficacy in vivo.

Example 2 In Vivo Inhibition of the Biological Activity of Human IL-1βusing IL-1β-Specific Antibodies, as Measured by the Impact on IL-1βStimulated Release of IL-6

To confirm the in vivo efficacy of AB7, its ability to block thebiological activity of human IL-1β was tested in mice. Details of theassay are described in Economides et al., Nature Med., 9: 47-52 (2003).Briefly, male C57/B16 mice (Jackson Laboratory Bar Harbor, Me.) wereinjected intraperitoneally with titrated doses of AB7, another IL-1βantibody, AB5, or a control antibody. Twenty-four hours after antibodyinjection, mice were injected subcutaneously with recombinant humanIL-1β (rhIL-1β) (from PeproTech Inc., Rocky Hill, N.J.) at a dose of 1μg/kg. Two hours post-rhIL-1β injection (peak IL-6 response time), micewere sacrificed, and blood was collected and processed for serum. SerumIL-6 levels were assayed by ELISA (BD Pharmingen, Franklin Lakes, N.J.)according to the manufacturer's protocol. Percent inhibition wascalculated from the ratio of IL-6 detected in experimental animal serumto IL-6 detected in control animal serum (multiplied by 100).

The results are set forth in FIG. 2A. The ability to inhibit the in vivoactivity of IL-1β was assessed as a function of IL-1β stimulated IL-6levels in serum. As illustrated by FIG. 2A, the AB7 and AB5 antibodieswere effective for inhibiting the in vivo activity of human IL-1β. Theseresults also show that a single injection of AB7 or AB5 can block thesystemic action to IL-1β stimulation and that such antibodies are usefulfor the inhibition of IL-1β activity in vivo.

A similar experiment was performed to further demonstrate the ability ofAB7 to neutralize mouse IL-1β in vivo, to support the use of thisantibody in mouse models of disease. It was determined that AB7 has anaffinity for human IL-1β that is ˜10,000 times greater than the affinityfor mouse IL-1β, and an in vitro potency in the D10.G4.1 assay that is˜1,000 times greater than that for mouse IL-1β. In the C57BL/6 mousemodel with IL-6 readout, the mice were injected with AB7 (3 or 300 ug)or PBS vehicle control i.p. 24 hours before a s.c. injection of human(FIG. 2B, panel A) or mouse (FIG. 2B, panel B) IL-1β (20 ng). Blood wasdrawn 2 hours later and serum samples were analyzed for IL-6 levels viaELISA. These data show maximum suppression of IL-6 levels (˜75%) inducedby human IL-1β at 3 μg (panel A), whereas submaximum suppression of IL-6levels (−50%) induced by mouse IL-1β was demonstrated with 300 μg (panelB). These results are consistent with the observation of far greateraffinity and in vitro potency of the AB7 antibody for human IL-1β, ascompared to mouse IL-1β. In addition, the data indicate that thisantibody may be used for mouse in vivo disease models with anappropriate higher dose than would be needed for treatment of humansubjects, where the antibody has far superior affinity and potency. Inthe case of other IL-1β antibodies, such as for example antibodiesdisclosed and/or cited herein, that do not exhibit such a property ofsignificantly higher affinity and in vitro potency for human IL-1β ascompared to mouse IL-1β, similar higher doses in mouse models may not benecessary.

Example 3 Pharmacokinetics of an Anti-IL-1β Antibody FollowingAdministration of a Single Intravenous or Subcutaneous Dose to Rats

To examine the pharmacokinetic profile, an IL-1β antibody designated AB7was administered to adult male rats as an intravenous (IV) bolus intothe tail vein at doses of 0.1, 1.0, or 10 mg/kg (Groups 1, 2, and 3respectively) or a subcutaneous (SC) dose between the shoulder blades at1.0 mg/kg (Group 4). Blood samples were collected via the jugular veincannula or the retro-orbital sinus at specified times for up to 91 daysafter dosing. Blood samples were centrifuged to obtain serum. Sampleswere analyzed for the concentration of anti-IL-1β antibody using analkaline phosphatase-based ELISA assay as follows.

IL-1β (Preprotech) was diluted to 0.5 μg/mL in PBS and 50 μL of thissolution was added to wells of Nunc-Immuno Maxisorp microtiter plates(VWR) and incubated overnight at 2-8° C. The antigen solution wasremoved and 200 μL of blocking buffer [1% bovine serum albumin (BSA) in1×PBS containing 0.05% Tween 20] was added to all wells and incubatedfor 1 hour at room temperature. After blocking, the wells were washedthree times with wash buffer (1×PBS, containing 0.05% Tween 20).Standards, samples and controls were diluted in sample diluent (25% RatSerum in 1×PBS containing 1% BSA and 0.05% Tween 20). Anti-IL-1βantibody standard solutions were prepared as serial two-fold dilutionsfrom 2000 to 0.24 ng/mL. Each replicate and dilution of the standards,samples and controls (50 μL) were transferred to the blocked microtiterplates and incubated for 1 hour at 37° C. After incubation, the wellswere washed 3 times with wash buffer. Alkaline phosphatase conjugatedgoat anti-human IgG (H+L) antibody (Southern Biotech Associates Inc,Birmingham, Ala.) was diluted 1/1000 in conjugate diluent (1% BSA in1×PBS containing 0.05% Tween 20). Fifty μL of the diluted conjugate wasadded to all wells except for the BLANK wells, which received 50 μL ofconjugate diluent only. The plates were incubated for 1 hour at 37° C.and then all wells were washed 3 times with wash buffer and 3 times withdeionized water. The substrate p-nitrophenylphosphate (1 mg/mL in 10%diethanolamine buffer, pH 9.8) was added to all wells and colordevelopment was allowed to proceed for 1 hour at room temperature, afterwhich 50 μL of 1 N NaOH was added to stop the reaction. The absorbanceat 405 nm was determined using a SPECTRAmax M2 Plate Reader (MolecularDevices, Menlo Park, Calif.) and a standard curve was then plotted asA₄₀₅ versus ng/mL of antibody standard. A regression analysis wasperformed and concentrations were determined for samples and controls byinterpolation from the standard curve. The limit of quantification was40 ng/mL.

As shown in FIG. 3, serum concentrations declined bi-exponentially amongthe IV dose groups. A compartmental analysis was performed on theindividual animal data, and resulting pharmacokinetic parameters wereaveraged for each dose group excluding those animals in which a RAHAresponse was generated. The serum levels of anti-IL-1β antibody declinedwith an average alpha phase half-life of 0.189±0.094 to 0.429±0.043 days(4.54 to 10.3 hours) and a beta phase half-life of 9.68±0.70 to 14.5±1.7days. Among rats receiving a 1 mg/kg subcutaneous dose of AB7 serumlevels increased to a peak of 4.26±0.065 μg/mL by 2-3 days, and declinedwith a half-life of 2.59±0.25 days.

Example 4 Pharmacokinetics of an Anti-IL-1β Antibody FollowingAdministration of a Single Intravenous Dose to Cynomolgus Monkeys

Adult male and female cynomolgus monkeys received the anti-IL-43antibody designated AB7 as an intravenous (IV) single bolus injection atdoses of 0.3, 3.0, or 30 mg/kg. Blood samples were collected fromanimals prior to dose, 5 minutes, 4 and 8 hours post dose on Day 1, andDays 2, 4, 8, 11, 15, 22, 29, 43 and 56. Samples were analyzed for theconcentration of anti-IL-1β antibody using an alkaline phosphatase-basedELISA assay as follows.

IL-1β solution was diluted to 0.5 μg/mL in PBS and 50 μl, of thissolution was added to wells of Nunc-Immuno Maxisorp microtiter plates(VWR) and incubated overnight at 2-8° C. The antigen solution wasremoved and 200 μL of blocking buffer [1% bovine serum albumin (BSA) in1×PBS containing 0.05% Tween 20] was added to all wells and thenincubated for 1-4 hours at room temperature. After blocking, the wellsof each plate were washed three times with wash buffer (1×PBS,containing 0.05% Tween 20). Standards, samples, and controls werediluted in sample diluent (2% Normal Cynomolgus Serum (NCS) in 1×PBScontaining 1% BSA and 0.05% Tween 20). Anti-IL-1β standard solutionswere prepared as serial two-fold dilutions from 8000 ng/mL. Eachreplicate and dilution of the standards, samples, and controls (50 μL)were transferred to the blocked microtiter plates and incubated for 1hour at 37° C. After the primary incubation, the wells were washed 3times with wash buffer and 50 μL of biotinylated rhIL-1 beta was addedto all wells. The plates were then incubated for 1 hour at 37° C. Thewells were washed 3 times with wash buffer and a tertiary incubationwith fifty μL of diluted alkaline phosphatase conjugated streptavidinwas added to all wells except for the BLANK wells, which received 50 μLof diluent only. The plates were incubated for 30 minutes at 37° C., andthen all wells were washed 3 times with wash buffer and 3 times withdeionized water. The substrate p-nitrophenylphosphate (1 mg/mL in 10%diethanolamine buffer, pH 9.8) was added to all wells. Color developmentwas allowed to proceed in the dark for 1 hour at room temperature, afterwhich 50 μL of 1 N NaOH was added to stop the reaction. The absorbanceat 405 nm was determined for all wells using a SPECTRAmax M2 PlateReader (Molecular Devices, Menlo Park, Calif.). A standard curve wasthen plotted as A₄₀₅ versus ng/mL of anti-IL-1β standard. A 4-parameterregression analysis was performed and concentrations were determined forsamples and controls by interpolation from the standard curve. The limitof quantification was 40 ng/mL.

For the single dose 0.3 and 3 mg/kg groups, the serum anti-IL-1βantibody levels declined with an average alpha phase half-life of9.40±2.00 hours, followed by a beta phase half-life of 13.3±1.0 days(FIG. 5). In cynomolgus monkeys receiving a single IV injection of 30mg/kg, serum levels of antibody declined more rapidly, with alpha phasehalf life of 10.9±3.2 hours, followed by a beta phase half-life of7.54±1.79 days. Modeling of plasma concentration-time profiles of 0.1,0.3, 1 and 10 mg/kg doses administered at five monthly intervals alsowas performed and is shown in FIG. 5.

Example 5 Effect of Anti-IL-1β Antibodies in a Human Islet Cell AssaySystem

As an in vitro model, human islet cells are isolated and then treatedwith high glucose levels to mimic the Type 2 diabetic environment.Anti-IL-1β antibodies may be used in the islet cell system to examinethe effect on beta cell function (insulin release in response toglucose), beta cell proliferation and apoptosis.

Islets are isolated from the pancreases of multiple human organ donorswith no history of diabetes or metabolic disease as described (Linetskyet al., Diabetes 46:1120-1123, 1997; Oberholzer et al., Transplantation69:1115-1123, 2000; Ricordi et al., Diabetes 37:413-420, 1988, Maedleret al., Proc. Natl. Acad. Sci. USA 101:8138-8143, 2004; WO2004/0002512). The islets are then cultured on extracellularmatrix-coated plates derived from bovine corneal endothelial cells(Novamed Ltd, Jerusalem), allowing the cells to attach to the plates andpreserving their functional integrity. The islets are cultured in CMRL1066 medium containing 100 U/mL penicillin, 100 ug/mL streptomycin and10% fetal bovine serum (GIBCO, Gaithersburg, Md.). To stimulate insulinsecretion, the culture medium is replaced with culture medium furthersupplemented with 5, 11 or 33 mM glucose, with or without addition offatty acid.

To measure insulin release in response glucose, islet cells are washedand pre-incubated for 30 minutes in Krebs-Ringer bicarbonate buffer(KRB) containing 3.3 mM glucose and 0.5% BSA. KRB is then replaced byKRB 3.3 mM glucose for 1 hr, which is then followed by an additional 1hr in KRB 16.7 mM glucose. Islet cells are extracted with 0.18 M HCl in70% ethanol for determination of insulin content using a human insulinRIA kit (CIS Biointernational, Gif-sur-Yvette, France). Beta cellapoptosis may be measured by a variety of methods. For example, cellsare double stained by the standard terminal deoxynucleotidyltransferase-mediated dUTP nic-end labeling (TUNEL) technique and alsofor insulin. In parallel, apoptosis also is confirmed by detection ofcaspase 3 activation or Fas expression as described (see for example, WO2004/002512; Maedler et al., 2004, ibid).

Example 6 Effect of Anti-IL-1β Antibodies in a Rat Pseudo Islet CellAssay System

Alternatively or in addition to the human islet cell model, rat pseudoislet cells may be used as an in vitro model to evaluate the effects ofanti-IL-1β antibodies. For example, pseudo islets may be prepared andtested as described in US 20060094714. Pancreata from four SpragueDawley rats are divided into small pieces, rinsed three times withHanks-Hepes buffer, and digested with collagenase (Liberase, 0.25 mg/ml,Roche Diagnostic Corp., Indianapolis, Ind., USA) at 37° C. in a waterbath shaker for 10 minutes. The digested pancreata tissue is then rinsedthree times with 50 ml of Hanks-Hepes buffer to remove the collagenaseand the tissue pellet is then filtered through a 250 micron filter. Thefiltrate is mixed with 16 ml of 27% Ficoll (Sigma, St. Louis, Mo., USA)w/v in Hanks-Hepes buffer and then centrifuged in a Ficoll gradient(23%, 20.5%, and 11%, respectively; 8 ml of each concentration) at 1,600rpm for 10 minutes at room temperature. The pancreatic islets areconcentrated at the interphase between 11% and 20.5%, and between 20.5%and 23% depending on the size of islets. The islets are collected fromthe two interphases, rinsed twice with calcium-free Hanks-Hepes buffer,and then suspended in 5 ml calcium-free Hanks-Hepes buffer containing 1mM EDTA and incubated for 8 minutes at room temperature. Trypsin andDNAse I are added to the islet suspension for a final concentration of25 ug/ml and 2 ug/ml, respectively, and the suspension is incubated withshaking at 30° C. for 10 minutes. The trypsin digestion is stopped byadding 40 ml RPMI 1640 (GIBCO Life Technologies, Invitrogen, Carlsbad,Calif.) with 10% FBS. The trypsin digested islet cells are then filteredthrough a 63 micron nylon filter (PGC Scientific, Frederick, Md.) toremove large cell clusters. The dispersed islet cells are then washed,counted, and seeded into “V-bottom” 96-well plates (2,500 cells perwell). The dispersed islet cell suspension is then centrifuged at 1,000rpm for 5 minutes. The Hanks-Hepes buffer is removed and replaced with200 ul RPMI 1640 medium containing 10% FBS, 1% Penicillin—Streptomycin,and 2 mM L-glutamine. Next, the 96-well plates are centrifuged at 1,000rpm for 5 minutes to collect the dispersed islet cells concentrated atthe V-bottom of the plate forming pseudo islets. These pseudo islets arethen cultured overnight in a cell culture incubator at 37° C. with 5%CO₂, and then used for assays.

Example 7 Effect of an IL-1β Antibody on Insulin Sensitivity in anAnimal Model

In vivo efficacy of an IL-1β antibody as an insulin-sensitizing agentmay be measured as the insulin and glucose-lowering activity of theantibody in a dietary model of insulin resistance. Male Sprague-Dawleyrats are placed on a high fat, high carbohydrate diet, containing 60%fructose, 10% lard, and 0.06% magnesium at 6 weeks of age. Two daysafter starting the diet, the rats are randomized into different groupsbased on antibody dose levels (ranging from 0.1 to 5 mg/kg body weight),route of administration (subcutaneous, intravenous, or intraperitonealroutes), and frequency of administration (daily to bi-weekly). Controlgroups receive either buffer (vehicle) only or an irrelevant antibody.Food and fluid intakes are measured each day and a pair-feeding protocolis utilized to insure equivalent food intakes among the 3 groups. After5 weeks, serum levels of glucose, insulin, and triglycerides areobtained in the semi-fasting state (the night before blood draw, animalsare given a restricted amount of food) and blood is drawn the followingmorning. The protocol is continued for an additional 9 weeks at whichtime glucose tolerance testing (OGTT) is performed in conscious animalsin the semi-fasted state by sampling blood for glucose and insulinmeasurements after oral administration of a glucose load (100 mg/100gram body weight). Serum levels of glucose and triglycerides aremeasured by spectrophotometric methods and insulin levels are measuredby radioimmunoassay (Linco, St. Louis, Mo.).

Example 8 IL-1β Antibodies for Treatment in a Psammomys obesus AnimalModel of T2D

The therapeutic effectiveness of an IL-1β antibody for preventing thedecline of β-cell mass observed in Type 2 diabetes patients is evaluatedin the gerbil Psammomys obesus, which shows insulin resistance anddevelops diet-induced obesity-linked diabetes, initially associated withhyperinsulinemia, and gradually progressing to severe hyperglycemia,accompanied by a transient increase in beta-cell proliferative activityand by a prolonged increase in the rate of beta-cell death, withdisruption of islet architecture (Donath et al., Diabetes 48:738-744,1999). To determine the effect of IL-16 antibody onhyperglycemia-induced beta-cell apoptosis and impaired proliferation inpancreatic islets of Psammomys obesus during development of diabetes,antibody is administered to the diabetes-prone animals (switched to ahigh energy diet) at multiple dose levels ranging from 0.1 to 5 mg/kgbody weight by the subcutaneous, intravenous, or intraperitoneal routes,with the antibody administrations repeated at intervals ranging fromdaily to weekly. Control groups of animals are either maintained on alow energy diet or switched to a high energy diet and treated withbuffer (vehicle) only or an irrelevant antibody. Subgroups of animalsare sacrificed on days 4, 7, 14, 21, and 28, whereupon blood iscollected and used to determine plasma glucose, insulin andtriglyerides. The pancreas is also removed, with a portion frozen at −70C for later determination of insulin content and the remaining portionfixed in 10% phosphate buffered formalin, embedded in paraffin andsectioned for analysis of Fas, IL-1β and insulin expression, andbeta-cell proliferation and apoptosis. Such analysis will allowdetermination of the prevention or delay in diabetes onset, protectionfrom hyperglycemia-induced beta-cell apoptosis, impaired proliferationand decreased β-cell mass, and normalization of pancreatic insulincontent.

Example 9 Use of an IL-1β Antibody in the Treatment of T2D in Humans

IL-1β antibodies or fragments described herein may be administered to ahuman patient in accordance with the invention for therapeutic treatmentand/or prevention of Type 2 diabetes. Specifically, in one example, anIL-1β antibody having the aforementioned properties (AB7, describedabove) is used for the therapeutic treatment of patients displayingsigns and symptoms of Type 2 diabetes. More specifically, safety andeffectiveness of an IL-1β antibody for Type 2 diabetes are demonstratedin one or more human clinical studies, including for example a trial ofthe following design.

A double-blind, placebo controlled human clinical study is performed inType 2 diabetes patients. Patients who meet inclusion criteria for thisstudy according to the American Diabetes Association (ADA) diagnosticcriteria for T2D:

-   -   Fasting blood glucose concentration≧126 mg/dL (≧7.0 mmol/L)        (must be measured within 28 days prior to Day 0)

OR

-   -   Symptoms of hyperglycemia (e.g., thirst, polyuria, weight loss,        visual blurring) AND a casual/random plasma glucose value of        ≧200 mg/dL (≧11.1 mmol/L) (must be measured within 28 days prior        to Day 0),        and with an HbA1c>7.5% and ≦12% (DCCT standard), are enrolled in        the study sequentially by study group and within each group are        randomly assigned to receive the IL-1β antibody or placebo. To        minimize risk to subjects, safety and tolerability are reviewed        at each dose level prior to escalating to the next dose level.        The treatment groups and numbers of subjects for the study are        shown in the following table:

Antibody Placebo Group Route # Subjects Dose # Subjects 1 IV or SC 50.01 mg/kg  1 2 IV or SC 5 0.03 mg/kg  1 3 IV or SC 5 0.1 mg/kg 1 4 IVor SC 5 0.3 mg/kg 1 5 IV or SC 5 1.0 mg/kg 1 6 IV or SC 5 3.0 mg/kg 1

On study Day 1, antibody or placebo is administered eithersubcutaneously or via a 30 minute constant rate intravenous infusion.Safety assessments, including the recording of adverse events, physicalexaminations, vital signs, clinical laboratory tests (e.g., bloodchemistry, hematology, urinalysis), plasma cytokine levels, andelectrocardiograms (ECGs) are conducted using standard medical practicesknown in the art. Blood samples are collected pre-dose administrationand at multiple time periods (e.g., days) post-administration to assessHbA1c, lipid profile including free fatty acids, HDL and LDLcholesterol, IL-1β antibody levels (pharmacokinetics), anti-IL-1βantibody responses, cytokine (e.g., IL-1β, IL-6, TNFα) levels, CRP,sodium, potassium, creatinine, AST, ALT and hematogram. Assays may alsobe performed for other cytokines and lymphokines, such as for example,those described herein. Additional blood samples may be collected atlater days than initially designed in those instances when levels of theadministered IL-1β antibody have not fallen below the limit ofdetection. Study assessments are conducted at specified timespost-treatment.

Clinical monitoring of treatment for Type 2 diabetes is performed basedon a primary efficacy endpoint of improvement in hemoglobin A1c (HbA1c,see for example Reynolds et al., BMJ, 333(7568):586-589, 2006).Improvement in HbA1c level is indicative of therapeutic efficacy of theanti-IL-1b treatment and generally should result in a decrease at leastabout of 0.5% or more. One or more of the following secondary endpointsare also determined to assess efficacy of the treatment for Type 2diabetes, such as for example fasting blood sugar (e.g., glucose) levels(e.g., ≦130, ≦120), 120 minute oral glucose tolerance test (OGTT),glucose/insulin C-peptide AUC (e.g., >50%, >60% increase), reduction indiabetes medication (e.g., insulin, oral hypoglycemic agent),improvement in insulin sensitivity, serum cytokine levels (e.g.,normalization), CRP levels, quality of life measurements, BMIimprovement (reduction 1%, 3%, 5%), pharmacokinetics, and the like(Saudek, et al., JAMA, 295:1688-97, 2006; Pfutzner et al., DiabetesTechnol Ther. 8:28-36, 2006; Norberg, et al., J Intern Med. 260:263-71,2006). Additional lipid profile analysis of samples includes thefollowing tests performed according to standard accepted methods knownin the art.

Test Method Lipoprotein electrophoresis Gel Electrophoresis Serumapolipoprotein A-I (apoA-I) Nephelometry Serum apolipoprotein A-II (apoA-II) Nephelometry Serum apolipoprotein B-48 (apo B-48) ELISA Serumapolipoprotein B-100 (apo B-100) Nephelometry Serum apolipoprotein Cs(apo Cs) Immunoturbidimetry for Apo CII and ApoCIII Serum apolipoproteinE (apo E) Nephelometry Serum apolipoprotein J (apo J) ELISA Serumamyloid A Nephelometry Plasma free fatty acids (FFA) Colorimetry Plasmaglycerol Colorimetry Serum LCAT ELISA Serum cholesteryl ester transferprotein (CETP) ELISA Serum hepatic lipase (HL) Fluorometry Serumparaoxonase 1 (PON1) UV/colorimetryPharmacokinetic Analysis

Samples are obtained for pharmacokinetic analysis at days 0, 1, 2, 3, 4,7, 9±1, 11±1, 14±1, 21±2, 28±2, 42±3, and 56±3. Preliminary analysis ofthe pharmacokinetics of AB7 in Type 2 diabetes subjects receiving asingle IV dose of 0.01 mg/kg showed serum concentration-time profileswith a terminal half-life of 22 days, clearance of 2.9 mL/day/kg andvolume of distribution of the central compartment of 50 mL/kg, verysimilar to serum volume (FIG. 7).

Blood Glucose and HbA1c Analysis

Samples are obtained and blood glucose measured at days 0, 7, 14±1,21±2, 28±2, 42±3, and 56±3, as well as the screening day. Assessment ofthese samples for a decrease in blood glucose levels is shown below forsamples from the first two dose cohorts (upper data line for eachsubject). Samples are obtained and HbA1c measured at days 0, 28±2, 42±3,and 56±3, as well as the screening day. Assessment of these samples fora decrease in HbA1c levels is shown below for samples from the first twodose cohorts (lower data line for each subject).

Cohort at 0.01 mg/kg dose

Day 0 Day 7 Day 14 Day 21 Day 28 Day 42 Day 56 Subject Screen Labs LabsLabs Labs Labs Labs Labs 5 200.00 247.00 212.00 179.00 213.00 242.00235.00 200.00 8.40 8.60 8.50 7.10 9.30 1 211.00 232.00 235.00 236.00199.00 221.00 252.00 204.00 9.30 9.60 9.50 9.10 9.80 2 229.00 131.00160.00 191.00 193.00 224.00 204.00 207.00 9.00 8.80 8.40 8.60 9.00 11290.00 283.00 300.00 177.00 308.00 278.00 292.00 302.00 11.80 11.6011.40 11.20 11.80 3 175.00 158.00 175.00 154.00 154.00 162.00 183.00170.00 8.20 8.20 7.70 7.80 8.10 4 238.00 255.00 270.00 275.00 289.00278.00 255.00 245.00 8.50 9.40 10.50 10.60 10.40Cohort at 0.03 mg/kg dose

Day 0 Day 7 Day 14 Day 21 Day 28 Day 42 Day 56 Subject Screen Labs LabsLabs Labs Labs Labs Labs 6 222.00 164.00 148.00 166.00 162.00 145.00207.00 8.40 8.40 8.20 8.40 7 208.00 109.00 101.00 120.00 81.00 108.00113.00 8.00 7.50 6.70 6.40 8 364.00 287.00 289.00 260.00 237.00 12.4012.00 9 204.00 128.00 124.00 117.00 112.00 113.00 7.90 7.50 7.10 12275.00 235.00 250.00 126.00 168.00 9.90 10.30 10 332.00 398.00 243.00187.00 220.00 11.50 11.50These data indicate the lower boundaries of a dose of IL-1β antibody asprovided herein, useful to achieve a therapeutic effect (e.g., decreasein glucose and/or HbA1c levels) in a subject following a singleadministration of antibody.C-Reactive Protein Analysis

C-reactive protein (CRP), which is released by the liver in response tovarious stress triggers, including IL-6, produced in response to IL-1,was also measured in serum at the same time points as the PK samples. APK/PD model was developed that incorporated a two compartment model forserum antibody level, and a concentration-dependent indirect response ofantibody on the rate of CRP production, with a linear rate ofelimination of CRP. After a single IV dose of 0.01 mg/kg antibody inType 2 diabetes subjects, CRP declined within 7-10 days to 66±22%relative to 100% pre-dose, based on the model fits (FIG. 8). After asingle IV dose of 0.03 mg/kg antibody in Type 2 diabetes subjects, CRPdeclined within 7-10 days to 40±12% relative to 100% pre-dose (FIG. 9).Data for the placebo controls are shown in FIG. 10. Based on these dataand on the model projections, the anticipated maintained CRP levelsfollowing monthly injections of antibody are approximated to be 40% at0.03 mg/kg, 16.5% at 0.1 mg/kg, 6.2% at 0.3 mg/kg, 1.9% at 1 mg/kg, and0.66% at 3 mg/kg per month (FIG. 11). These data indicate that an IL-1βantibody as provided herein may be administered as infrequently as onemonth or longer to achieve a therapeutic effect (e.g., decrease in CRPlevels) in a subject following a single administration of antibody.

Based on results obtained from the first clinical study, additionalclinical trials are performed. Such trials may include one or more ofthe above dosages, as well as or alternatively one or more other dosagesof IL-1β antibody, longer treatment and/or observation periods andgreater numbers of patients per group (at least about 10, 50, 100, 200,300, 400, 500, 750, 1000), in accordance with the invention. Inaddition, these and other studies also may be used to determine theperiod of time required to reach a desired therapeutic benefit based onchange of a specific parameter (e.g., decrease in blood sugar, decreasein HbA1c, decrease in CRP), as well as the duration of the desiredtherapeutic benefit based on change of a specific parameter (e.g.,decrease in blood sugar, decrease in HbA1c, decrease in CRP), beforeadditional dosages are administered.

Example 10 Effect of IL-1β Antibody on Adipocyte Function and InsulinResistance

An in vitro assay using cultured adipocytes may be used to demonstrate areduction (e.g., blocking) of IL-1β-induced insulin resistance by usinganti-IL-1β antibodies. The 3T3-L1 preadipocyte cell line obtained fromATCC (# CL-173) is grown at 7% CO₂ and 37° C. in DMEM, 25 mM glucose,and 10% calf serum and induced to differentiate in adipocytes. Briefly,2 days after confluence, medium is exchanged for DMEM, 25 mM glucose,and 10% FCS supplemented with isobutylmethylxanthine (0.25 mM),dexamethasone (0.25 μM), insulin (5 μg/ml), and pioglitazone (10 μM).The medium is removed after 2 days and replaced with DMEM, 25 mMglucose, and 10% FCS supplemented with insulin (5 μg/ml) andpioglitazone (10 μM) for 2 days. Then the cells are fed every 2 dayswith DMEM, 25 mM glucose, and 10% FCS. 3T3-L1 adipocytes are used 8-15days after the beginning of the differentiation protocol.

Human preadipocytes (Biopredic International, Rennes, France) are grownat 5% CO₂ and 37° C. in DMEM Ham's F12 containing 15 mM HEPES, 2 mML-glutamine, 5% FCS, 1% antimycotic solution, ECGS/H-2, hEGF-5, andHC-500 from supplement pack preadipocyte growth medium (Promocell,Heidelberg, Germany). Differentiation into adipocytes is induced afterconfluence by exchanging the medium for DMEM Ham's F12 15 mM HEPES, 2 mML-glutamine, and 3% FCS supplemented with biotin (33 μM), insulin (100nM), pantothenate (17 μM), isobutylmethylxanthine (0.2 mM),dexamethasone (1 μM), and rosiglitazone (10 μM). The medium is removedafter 3 days and replaced with Ham's F12 containing 15 mM HEPES, 2 mML-glutamine, and 10% FCS supplemented with biotin (33 μM), insulin (100nM), pantothenate (17 μM), and dexamethasone (1 μM). Then the cells arefed every 2 days with the same medium. Human adipocytes are used 15 daysafter the beginning of the differentiation protocol. Human preadipocytesalso may be obtained from alternative sources, such as for example celllines XA15A1 and XM18B1 (Lonza, Allendale, N.J.).

The role of IL-1β in inducing insulin resistance (decrease insulinsensitivity) in cultured adipocytes is shown by incubating adipocyteswith IL-1 μl (e.g., 20 ng/mL, 48 hrs) and then incubating with differentconcentrations of insulin (e.g., 0.5 nM, 100 nM; 20 min), followed bymeasurement of glucose transport after the addition of2-[³H]deoxyglucose. Insulin resistance is determined as a reduction inglucose uptake, and the effect of an anti-IL-1β antibody at reducing(e.g., blocking) insulin resistance is readily measured in thisadipocyte cell culture system.

The role of IL-1β in directly stimulating the production of adipokinesand cytokines by adipoyctes (e.g., leptin, resistin, visfatin, IL-6,MCP-1 (CCL2), RANTES, PAI-1, Acylation-stimulating protein, SAA3,Pentraxin-3, macrophage migration inhibition factor, IL-1RA, IL-12,IL-8, IL-6, TNF-α) is determined by culturing adipoyctes in the absenceor presence of different concentrations of IL-1β for different lengthsof time as described above, and measuring levels of adipocytes andcytokines in the culture-conditioned medium, usually via ELISA or othercommonly used methods. In addition, the effects of treatingIL-1β-stimulated adipocyte cultures with an anti-IL-1β neutralizingantibody on the induction of adipokine and/or cytokine secretion relatedto insulin resistance are measured. Similarly, the effects of treatingwith an anti-IL-1β antibody on suppressing the insulin-sensitizingadipokine, adiponectin, are measured. To address whether anti-IL-1βantibody treatment neutralizes the effects of endogenously-produced IL-1derived from immune/inflammatory cells (e.g., macrophages) duringadipose tissue inflammation, different numbers of human macrophages(monocyte-derived or various monocytic cell lines) are cultured with theadipocyte cultures described above in the absence or presence ofanti-IL-1β antibody and modulation of adipokines and cytokines aremeasured. In addition, the modulating effects on adipokine and cytokinesecretion after in vivo treatment of a subject with an anti-IL-1βantibody are measured in the circulation (e.g., serum, plasma), as ameans to demonstrate efficacy.

Example 11 Inhibition of Cytokine Production in Human Whole Blood by anIL-1β Antibody

Measuring cytokines in blood during a disease or the treatment of adisease can be useful for determining disease severity or response to atherapy. Usually, cytokine levels are measured in serum, but this methoddoes not necessarily measure total cytokines. Many cytokines can beinside cells (intracellular). In addition, the ability for a cell toproduce a cytokine may be more useful information than the level ofcirculating cytokine.

A method of stimulating whole blood was used to determine cytokineproduction and the effect of treating with an anti-IL-1β antibody. Bloodwas drawn from patients into sterile heparinized tubes and then 250 ulof the whole blood was added to each 4 mL orange top Corning sterilecryotube set up as follows:

Control Series

All tubes were pre-filled with 550 ul of RPMI. To tube 1 (control), 200ul RPMI was added and to tubes 2-10, 100 ul additional RPMI was added.To each of tubes 2-10, 100 ul of dilutions of an anti-IL-1β antibody(AB7) was added.

Test Series

A similar series of antibody dilutions was set up as detailed above.

All tubes were mixed well using a 10 second vortex. Control series tubesA1-10 then received an additional 100 ul of RPMI, were vortexed 10seconds, the screw cap tightly fixed and the tubes placed in incubator.To Test series tubes B1-10, 100 ul of heat-killed Staphylococcusepidermidis (final concentration of 1:1000 of stock resulting in abacterium:white blood cell ration of 10:1) was added, the tubes werethen vortexed for 10 seconds, capped and placed in 37° C. incubator.After 24 hours incubation, the cultures were all lysed with Triton X(0.5% final) to release the cell contents and the lysates were frozen.After lysis of the whole blood cultures, the tubes subjected to freezethaw cycles and cytokine levels are measured by standard cytokine ELISAassays for human TNFα, IL-6, IFNγ, IL-8, IL-1α, IL-1Ra and IL-1β (R&DSystems, Minneapolis, Minn.).

Cytokines measured in the control series tubes, which contain onlysterile culture medium and antibody (where indicated), reflect thespontaneous level of stimulation. In healthy subjects, very low levelsof the various cytokines are found when measured after 24 hours ofincubation. In patients with untreated diseases, the levels may behigher. The Test series of tubes additionally contained a defined amountof heat-killed Staphylococcus epidermidis, which stimulates productionof a number of cytokines. If the anti-IL-1β antibody treatment isefficacious, this will be reflected by reduces cytokine production.

As shown in FIG. 6, the high affinity anti-IL-1β antibody AB7 was veryeffective at inhibiting the production of IL-1β in human blood. In anaverage of three human samples, the antibody inhibited the production ofIL-1β induced by Staphylococcus epidermidis by 50% at 0.1 pM and by 75%at 3 pM. At 100 pM, inhibition was 100%. Interferon gamma (IFNγ) wasinduced by Staphylococcus epidermidis and AB7 reduced IFNγ induced byStaphylococcus epidermidis by 75% at 100 pM.

Example 12 Effect of Anti-IL-1β Antibody on Diabetes in the Non-ObeseDiabetic (NOD) Mouse

To demonstrate efficacy of an anti-IL-1β antibody in a mouse diabetesmodel, female 3- to 4-week-old NOD mice (Jackson Laboratories, BarHarbor, Me.) are obtained and housed in a vivarium under pathogen-freeconditions. Various doses of anti-IL-1β antibody (e.g., 3 to 600 μg) arediluted in a suitable vehicle (e.g., PBS) and administered inprediabetic female NOD mice starting no later than 6 weeks of age, usingdifferent routes (e.g., intraperitoneally, subcutaneously,intravenously) at defined intervals (e.g., weekly, biweekly, monthly).Blood glucose is monitored using a glucometer (Encore Glucometer; Bayer,Elkhart, Ind.) at weekly intervals, beginning at 10 weeks of age. Micewith blood glucose levels 200 mg/dl on two consecutive occasions areconsidered diabetic (diabetes onset is usually observed around 15 to 20weeks of age and the incidence usually achieves a maximum around 90% by30 weeks of age). The data are calculated as the percentage of animalsremaining diabetes-free over the course of the experiment. Thedifferences between curves are tested using the log-rank test, whichcompares the distributions over the entire observation period.

In another NOD mouse model, efficacy of the anti-IL-1b antibody isdemonstrated in a cyclophosphamide (CY)-accelerated disease model (Reddyet al., Histochem J. 33:317-327, 2001; Cailleau et al., Diabetes46:937-940, 1997; Reddy et al., Histochem J., 34:1-12, 2002; Harada etal., Diabetologia 27:604-606, 1984; Nicoletti, et al., Eur J Immunol24:1843-7, 1994). Non-diabetic 4- to 8-week old male (or female) NODmice are obtained (Jackson Laboratories, Bar Harbor, Me.) and housed ina vivarium under pathogen-free conditions. Mice are injected with asingle dose of CY (Sigma) at 200 mg/Kg and are treated at variousaccelerated intervals due to the accelerated nature of the model (e.g.,once per week, twice per week) for two to three weeks with or withoutvarious doses of anti-IL-1β (e.g., 3 ug, 30 ug, 150 ug, 600 μg) orisotype control antibodies diluted in a suitable vehicle (e.g., PBS)using different routes of administration (e.g., intraperitoneally,subcutaneously, intravenously). Urine glucose (glycosuria) levels aremonitored three times a week and blood glucose levels are monitored onceper week using a glucometer beginning the day before CY injection. Urineglucose levels>20 mmol/L on two consecutive occasions are considereddiabetic and reduction of urine glucose levels by an anti-IL-1β antibodyare a measure of efficacy.

In another model, the efficacy of an anti-IL-1β antibody is evaluated ina diabetes-recurrence model of pancreatic islet transplantation (nottransplant rejection) (Mellgren et al., Diabetologia 29:670-2, 1986;Sandberg, et al., Clin Exp Immunol 108:314-7, 1997). Non-diabetic 4- to8-week old female NOD mice are obtained (Jackson Laboratories, BarHarbor, Me.) and housed in a vivarium under pathogen-free conditions.Pancreatic islets are prepared from 5-6 week-old non-diabetic male andfemale NOD mice before marked leukocytic infiltration and transplantedunder the kidney capsule of spontaneous diabetic (15 to 20 week-old)female NOD mice (400 to 450 islets/mouse). Transient normoglycemiaoccurs shortly after transplantation and hyperglycemia usuallyre-appears approximately 6 days after transplantation. Mice are treatedwith or without various doses of anti-IL-1β (e.g., 3 ug, 30 ug, 150 ug,600 μg) or isotype control antibodies diluted in a suitable vehicle(e.g., PBS) using different routes of administration (e.g.,intraperitoneally, subcutaneously, intravenously). Blood glucose levelsare monitored before and after transplantation once or twice per weekusing a glucometer and mice with levels>200 mg/d1 on two consecutiveoccasions are considered diabetic and reduction of blood glucose levelsby an anti-IL-1β antibody are a measure of efficacy.

Example 13 Treatment of Low-Dose Streptozotocin-Induced Diabetes Modelin C57BL/K Mice

To demonstrate efficacy of an anti-IL-1β antibody in a multiple low-dosestreptozotocin (STZ)-induced hyperglycemia and insulitis diabetes model(Sandberg, et al., Biochem Biophys Res Commun 202:543-548, 1994; Reddy,et al., Ann N Y Acad Sci 1079:109-113, 2006), 4- to 8-week old C57BL/Kmice are obtained (Jackson Laboratories, Bar Harbor, Me.) and housed ina vivarium under pathogen-free conditions. Mice receive five dailyinjections of STZ (40 mg/kg) in this accelerated model and are subjectedto accelerated treatment (starting one day before STZ injection) atvarious intervals (e.g., once, twice, or three times a week) for one tothree weeks with or without various doses of anti-IL-1β (e.g., 3 ug, 30ug, 150 ug, 600 ug) or isotype control antibodies diluted in a suitablevehicle (e.g., PBS) using different routes of administration (e.g.,intraperitoneally, subcutaneously, intravenously). Blood glucose levelsare monitored once per week using a glucometer beginning one day beforeSTZ injection. Blood glucose levels>200 mg/dl on two consecutiveoccasions are considered diabetic and reduction of blood glucose levelsby an anti-IL-1β antibody are a measure of efficacy.

Example 14 Treatment in the Diet-Induced Obesity Model of Type 2Diabetes

The efficacy of an anti-IL-1β antibody was tested in the diet-inducedobesity (DIO) model of Type 2 diabetes. In this model, mice fed a dietwith high fat content become obese over a period of several weeks, andthey exhibit impaired glucose tolerance and impaired insulin secretionwhen challenged with a bolus injection of glucose. C57BL/6 male mice, 6weeks of age, were fed normal diet (ND, Teklad, 5 kcal % fat) orSurwit's high fat, high sucrose diet (HFD, Research Diets #D12331, 58kcal % fat). Antibody dosing was initiated the day before. Theanti-IL-1β test antibody (AB7) and isotype control human IgG2 antibodywere administered by intraperitoneal (i.p.) injection. Antibodies weredosed twice a week for 4 weeks. Body weight was also recorded twice aweek. After 4 weeks, mice were subjected to a glucose tolerance test(GTT). In the GTT, mice are fasted overnight, and then injected i.p.with 1 g/kg of glucose. Blood glucose is measured from tail nicks at 0,15, 30, 60, 90, and 120 minutes after the injection, using a FreeStyleglucometer. FIG. 12 shows that mice fed a high fat diet for 4 weeks hadimpaired glucose tolerance compared to mice on the normal diet (FIG. 1).Administration of an IL-1β test antibody protected HFD mice fromimpaired glucose tolerance. At 60 minutes during the GTT, performance ofmice dosed with 1 mg/kg of IL-1β antibody were significantly better thanmice that had received IgG2 control antibody (*, p<0.05). Notably, thepositive results in this mouse model were observed even though the AB7antibody has a much lower affinity (10,000 fold) and in vitro potencyfor mouse IL-1β, as compared to human IL-1β, as described in Example 2above.

All references, including publications, patent applications, andpatents, cited herein are hereby incorporated by reference to the sameextent as if each reference were individually and specifically indicatedto be incorporated by reference and were set forth in its entiretyherein.

The use of the terms “a” and “an” and “the” and similar referents in thecontext of describing the invention (especially in the context of thefollowing claims) are to be construed to cover both the singular and theplural, unless otherwise indicated herein or clearly contradicted bycontext. The terms “comprising,” “having,” “including,” and “containing”are to be construed as open-ended terms (i.e., meaning “including, butnot limited to,”) unless otherwise noted. Wherever an open-ended term isused to describe a feature or element of the invention, it isspecifically contemplated that a closed-ended term can be used in placeof the open-ended term without departing from the spirit and scope ofthe invention. Recitation of ranges of values herein are merely intendedto serve as a shorthand method of referring individually to eachseparate value falling within the range, unless otherwise indicatedherein, and each separate value is incorporated into the specificationas if it were individually recited herein. All methods described hereincan be performed in any suitable order unless otherwise indicated hereinor otherwise clearly contradicted by context. The use of any and allexamples, or exemplary language (e.g., “such as”) provided herein, isintended merely to better illuminate the invention and does not pose alimitation on the scope of the invention unless otherwise claimed. Nolanguage in the specification should be construed as indicating anynon-claimed element as essential to the practice of the invention.

Preferred embodiments of this invention are described herein, includingthe best mode known to the inventors for carrying out the invention.Variations of those preferred embodiments may become apparent to thoseworking in the art upon reading the foregoing description. The inventorsexpect skilled artisans to employ such variations as appropriate, andthe inventors intend for the invention to be practiced otherwise than asspecifically described herein. Accordingly, this invention includes allmodifications and equivalents of the subject matter recited in theclaims appended hereto as permitted by applicable law. Moreover, anycombination of the above-described elements in all possible variationsthereof is encompassed by the invention unless otherwise indicatedherein or otherwise clearly contradicted by context.

The invention claimed is:
 1. A method of treating in a human, Type 2diabetes, or a Type 2 diabetes-induced disease or condition selectedfrom the group consisting of: insulin resistance, decreased insulinproduction, hyperglycemia, hyperinsulinemia, and obesity, the methodcomprising administering an anti-IL-1β antibody or fragment thereof tothe human, wherein the antibody or antibody fragment binds to humanIL-1β with a dissociation constant of about 250 pM or less.
 2. Themethod of claim 1, wherein the Type 2 diabetes-induced disease orcondition is decreased insulin production.
 3. The method of claim 1,wherein the Type 2 diabetes-induced disease or condition ishyperglycemia.
 4. The method of claim 1, wherein the Type 2diabetes-induced disease or condition is hyperinsulinemia.
 5. The methodof claim 1, wherein the Type 2 diabetes-induced disease or condition isobesity.
 6. The method of claim 1, wherein the Type 2 diabetes-induceddisease or condition is insulin resistance.
 7. The method of claim 1,wherein administration of an initial dose of the antibody or antibodyfragment is followed by the administration of one or more subsequentdoses.
 8. The method of claim 7, wherein administration of an initialdose of the antibody or antibody fragment is followed by theadministration of two or more subsequent doses.
 9. The method of claim1, wherein administration of an initial dose of the antibody or antibodyfragment is followed by the administration of one or more subsequentdoses, and wherein said one or more subsequent doses are in an amountthat is approximately the same or less than the initial dose.
 10. Themethod of claim 1, wherein administration of an initial dose of theantibody or antibody fragment is followed by the administration of oneor more subsequent doses, and wherein at least one of the subsequentdoses is in an amount that is more than the initial dose.
 11. The methodof claim 1, wherein the antibody or antibody fragment binds to humanIL-1β with a dissociation constant of about 10 pM or less.
 12. Themethod of claim 11, wherein the antibody or antibody fragment binds tohuman IL-1β with a dissociation constant of about 1 pM or less.
 13. Themethod of claim 12, wherein the antibody or antibody fragment binds tohuman IL-1β with a dissociation constant of about 0.5 pM or less. 14.The method of claim 7, wherein the initial dose and each one or moresubsequent doses are separated from each other by an interval of atleast about 2 weeks.
 15. The method of claim 14, wherein the initialdose and each one or more subsequent doses are separated from each otherby an interval of at least about 1 month.
 16. The method of claim 15,wherein the initial dose and each one or more subsequent doses areseparated from each other by an interval of at least about 3 months. 17.The method of claim 16, wherein the initial dose and each one or moresubsequent doses are separated from each other by an interval of atleast about 6 months.
 18. The method of claim 17, wherein the initialdose and each one or more subsequent doses are separated from each otherby an interval of at least about 12 months.
 19. The method of claim 1,wherein the antibody or antibody fragment is administered in one or moredoses of 3 mg/kg or less of antibody or fragment.
 20. The method ofclaim 19, wherein the antibody or antibody fragment is administered inone or more doses of 1 mg/kg or less of antibody or fragment.
 21. Themethod of claim 20, wherein the antibody or antibody fragment isadministered in one or more doses of 0.5 mg/kg or less of antibody orfragment.
 22. The method of claim 21, wherein the antibody or antibodyfragment is administered in one or more doses of 0.1 mg/kg or less ofantibody or fragment.
 23. The method of claim 22, wherein the antibodyor antibody fragment is administered in one or more doses of 0.03 mg/kgor less of antibody or fragment.
 24. The method of claim 23, wherein theantibody or antibody fragment is administered in one or more doses of0.01 mg/kg or less of antibody or fragment.
 25. The method of claim 19,wherein the one or more doses are at least 0.01 mg/kg of antibody orfragment.
 26. The method of claim 1, wherein the anti-IL-1β antibody orantibody fragment is a neutralizing antibody.
 27. The method of claim 1,wherein the anti-IL-1β antibody or antibody fragment binds to an IL-1βepitope such that the bound antibody or fragment substantially permitsthe binding of IL-1β to IL-1 receptor I (IL-1RI).
 28. The method ofclaim 1, wherein the antibody or antibody fragment does not detectablybind to IL-1α, IL-1R or IL-1Ra.
 29. The method of claim 1, wherein theantibody or antibody fragment binds to an epitope contained in thesequence ESVDPKNYPKKKMEKRFVFNKIE (SEQ ID NO: 1).
 30. The method of claim1, wherein the antibody or antibody fragment binds to an epitopeincorporating Glu64 of IL-1β.
 31. The method of claim 1, wherein theantibody or antibody fragment binds to amino acids 1-34 of the Nterminus of IL-1β.
 32. The method of claim 1, wherein the antibody orantibody fragment is human engineered or humanized.
 33. The method ofclaim 1, wherein the antibody or antibody fragment is human.
 34. Themethod of claim 1, wherein the anti-IL-1β antibody or antibody fragmentis administered by subcutaneous, intravenous or intramuscular injection.35. The method of claim 1, wherein the antibody or antibody fragment isadministered as a fixed dose, independent of a dose per subject weightratio.
 36. The method of claim 35, wherein the antibody or antibodyfragment is administered in one or more doses of 500 mg or less ofantibody or fragment.
 37. The method of claim 36, wherein the antibodyor antibody fragment is administered in one or more doses of 250 mg orless of antibody or fragment.
 38. The method of claim 37, wherein theantibody or antibody fragment is administered in one or more doses of100 mg or less of antibody or fragment.
 39. The method of claim 38,wherein the antibody or antibody fragment is administered in one or moredoses of 25 mg or less of antibody or fragment.
 40. The method of claim35, wherein the antibody or antibody fragment is administered in one ormore doses of at least 1 mg of antibody or fragment.
 41. The method ofclaim 35, wherein the antibody or antibody fragment is administered inone or more doses of about 10 mg to about 100 mg of antibody orfragment.
 42. The method of claim 1, wherein said method is inconjunction with at least one additional treatment method, saidadditional treatment method comprising administering at least onepharmaceutical composition comprising an active agent other than anIL-1β antibody or antibody fragment.
 43. The method of claim 1, whereinsaid method prevents or delays the need for at least one additionaltreatment method, said additional treatment method comprisingadministering at least one pharmaceutical composition comprising anactive agent other than an IL-1β antibody or antibody fragment.
 44. Themethod of claim 1, wherein said method reduces the amount, frequency orduration of at least one additional treatment method, said additionaltreatment method comprising administering at least one pharmaceuticalcomposition comprising an active agent other than an IL-1β antibody orantibody fragment.
 45. The method of claim 42, wherein said at least onepharmaceutical composition comprising an active agent other than anIL-1β antibody or antibody fragment is selected from the groupconsisting of a sulfonylurea, a meglitinide, a biguanide, analpha-glucosidase inhibitor, a thiazolidinedione, a glucagon-likepeptide, and insulin.
 46. The method of claim 45, wherein said activeagent is a sulfonylurea.
 47. The method of claim 45, wherein said activeagent is a meglitinide.
 48. The method of claim 45, wherein said activeagent is a biguanide.
 49. The method of claim 45, wherein said activeagent is an alpha-glucosidase inhibitor.
 50. The method of claim 45,wherein said active agent is a thiazolidinedione.
 51. The method ofclaim 45, wherein said active agent is a glucagon-like peptide.
 52. Themethod of claim 45, wherein said active agent is insulin.
 53. The methodof claim 1, wherein administration of an initial dose of the antibody orantibody fragment is followed by the administration of one or moresubsequent doses, and wherein the plasma concentration of said antibodyor antibody fragment in the human is maintained at a level of at leastabout 0.03 μg/ml during a course of treatment with said initial dose andone or more subsequent doses.
 54. The method of claim 53, wherein theplasma concentration of said antibody or antibody fragment is maintainedat a level of at least about 0.1 μg/ml during the course of treatment.55. The method of claim 54, wherein the plasma concentration of saidantibody or antibody fragment is maintained at a level of at least about0.3 μg/ml during the course of treatment.
 56. The method of claim 1,wherein the antibody or antibody fragment thereof has a lower IC₅₀ thanan IL-1β receptor antagonist in a human whole blood IL-1β inhibitionassay that measures IL-1β induced production of IL-8.